Systems and methods for characterization of molecules

ABSTRACT

The present invention generally provides systems and methods for the detection, identification, or characterization of differences between properties or behavior of corresponding species in two or more mixtures comprised of molecules, including biomolecules and/or molecules able to interact with biomolecules, using techniques such as partitioning. The experimental conditions established as distinguishing between the mixtures of the molecules using the systems and methods of the invention can also be used, in some cases, for further fractionation and/or characterization of the biomolecules and/or other molecules, using techniques such as single-step or multiple-step extraction, and/or by liquid-liquid partition chromatography. The methods could also be used for discovering and identifying markers associated with specific diagnostics, and can be used for screening for such markers once discovered and identified during diagnostics screening.

RELATED APPLICATIONS

This application claims priority to all of the following applicationsaccording to the following recitation of priority relationships. Thisapplication, is a continuation of U.S. patent application Ser. No.15/138,538, filed Apr. 26, 2016, entitled “Systems and Methods forCharacterization of Molecules,” by Chait, et al., which is acontinuation of U.S. patent application Ser. No. 14/049,249, filed Oct.9, 2013, entitled “Systems and Methods for Characterization ofMolecules,” by Chait, et al., which is a continuation of U.S. patentapplication Ser. No. 13/316,625, filed Dec. 12, 2011, entitled “Systemsand Methods for Characterization of Molecules,” by Chait, et al., whichis a continuation of U.S. patent application Ser. No. 11/818,911, filedJun. 15, 2007, entitled “Systems and Methods for Characterization ofMolecules,” by Chait, et al., which is a continuation-in-part of U.S.patent application Ser. No. 11/440,222, filed May 24, 2006, entitled“Systems and Methods for Characterization of Molecules,” by Chait, etal.; which application is a continuation-in-part of U.S. patentapplication Ser. No. 10/560,373, filed Dec. 12, 2005, entitled “Systemsand Methods for Characterization of Molecules,” by Chait, et al.; whichapplication claims priority to International Patent Application No.PCT/US04/019343, filed Jun. 14, 2004, entitled “Systems and Methods forCharacterization of Molecules,” by Chait, et al., published as WO2004/111655 on Dec. 23, 2004; which application claims priority to U.S.Provisional Patent Application Ser. No. 60/478,645, filed Jun. 12, 2003,entitled “Systems and Methods for Identifying and Using MolecularMarkers,” by Chait, et al.; and to U.S. Provisional Patent ApplicationSer. No. 60/561,945, filed Apr. 14, 2004, entitled “Systems and Methodsfor Characterization of Molecules,” by Chait, et al. Each of theseapplications is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention is generally related to the separation,fractionation, and/or characterization of molecules and/or biomoleculesin a mixture. More particularly, the invention is related to developingmethods for separation using, for example, aqueous multi-phasepartitioning, to discover differences between two or more mixtures ofmolecules or biomolecules, which can reflect structural and functionalcharacteristics of biomolecules and/or molecules which interact withbiomolecules. This invention is also related to discovering, selecting,and using markers for the purpose of classifying mixtures, where themarkers are species within the mixture that are different between themixtures.

BACKGROUND OF THE INVENTION

Many diseases and/or other pathological processes or conditions arecaused by dysfunction and/or disregulation of certain proteins. Thesedisease-related proteins may have their structures altered, relative totheir “normal” or “wild-type” counterparts and/or may be expressed inlarger (up-regulated expression) or lower (down-regulated expression)quantities in a given disease state, relative to “normal” physiologicalconditions. In some cases, proteins having altered structure and/orfunction may be used as protein markers associated with a particularhuman or animal disease, for instance, as a diagnostic for the earlierdetection of the disease, or the like. In many cases, the particularprotein(s) of relevance to a given pathological process of a disease orother condition are unknown. Identification of such protein(s) would beuseful for development of new diagnostic tests, or the like.

One general approach to the identification and characterization ofprotein markers is based on the analysis of protein compositions ofsamples of biological material (biological fluids, such as serum,plasma, and cerebrospinal fluid, tissues, cells, etc.) using highresolution separation techniques. For instance, proteins isolated fromcontrol and experimental populations can be subjected to proteolyticcleavage, and their cleavage products identified using liquidchromatography (LC) coupled with tandem mass spectrometry (LC-MS-MS).Many protein separation techniques are based on multi-dimensionalseparation of proteins from a sample, typically by two-dimensional gelelectrophoresis (2-DE) or two-dimensional high-performance liquidchromatography (2D-HPLC). The 2-D protein maps may be obtained andcompared for pathological samples with those for reference samples;positions of proteins observed as “spots” on 2-DE maps or as “peaks” on2D-HPLC maps can be compared and those that are present (or absent) inthe maps obtained from pathological samples but absent (or present) inthe maps obtained from the reference samples may be judged as likely tocorrespond to pathologically relevant proteins. Additionally, quantitiesof proteins estimated as intensities of the spots (or peaks) may beevaluated and compared between the pathological and reference samples.Those that are significantly different may be considered aspathologically relevant.

It has also been recently established that a pattern of thepresence/absence and/or the relative quantities of multiple proteins (a“signature”) may also be of diagnostic relevance, where the proteinsjudged to be of interest are identified by peptide mapping and massspectrometry. Mathematical or statistical techniques, such as patternrecognition techniques, could be used to analyze the pattern produced bythese experimental techniques and produce a diagnostic classification.However, this approach is often highly inefficient, for example, due tothe inherent necessity of analyzing all of the proteins in a givensample, whereas only a small portion of the proteins may have anypathological relevance.

Several different methods for reducing the analytical complexity ofprotein mixtures have been developed. These methods are typically basedon fractionation of the original mixture prior to 2-D analysis by gelelectrophoresis or 2-D HPLC. One such method is separation of proteinsby the technique of free-flow electrophoresis. However, this technique,while fractionating the original protein mixture, may result in multiple2-D analysis of simplified fractions, i.e. while reducing the complexityof analysis and improving resolution, it inherently increases the numberof samples for further analysis.

Another method is fractionation based on the affinity of proteins todifferent natural ligands and/or pharmacological compounds; however,this approach, while allowing separation of proteins according toprotein functions, may inherently result in an increase in the number ofsamples for further analysis, and often requires additional knowledge orpresumption concerning the differences between the samples.

A disadvantage of most present fractionation techniques is that theygenerally cannot preserve protein-protein or protein-ligandinteractions. Differences among biological interactions are oftenimportant for elucidating and detecting changes among samples.Additionally, most of the fractionation techniques today rely onseparation due to a fixed physical attribute, such as molecular size ornet charge. While these attributes are very important for distinguishingamong biomolecules in a complex mixture, they generally do not cover allof the potential differences between biomolecules representing, e.g.,normal vs. disease states, differences in configuration etc. Yet anotherimportant disadvantage of present fractionation techniques is related totheir inability to separate mixtures based on differences betweenstructural changes in, e.g., glycosylation patterns or conformationalchanges. These changes are often important for identifying proteins thateither participate in and/or are the result of a disease state. Forexample, if a protein is misfolded as a result of genetic mutation, theprotein's net charge and size are unlikely to vary significantly, andmore importantly, the protein's expression level might be the same forthe underlying normal vs. disease states. Finally, natural geneticvariability among individuals can significantly contribute to a verylarge scatter in the expression levels (concentration) of biomoleculesin a biological sample. This variability may necessitate the use ofstatistically large number of samples to robustly detect differencesinnate to a particular pathological condition rather than to geneticvariability. Natural genetic variability often is a significanthindrance in implementing protein marker based diagnostics by reducingsensitivity and/or specificity of the test. A technique that isinsensitive to the particular expression level of each biomolecule andinstead is sensitive to structural difference in that biomolecule ispotentially of great interest in the field.

SUMMARY OF THE INVENTION

The present invention generally relates to the analysis andcharacterization of biomolecules, complexes comprising biomolecules,molecules which interact with biomolecules and/or analogous speciesthereof. The results of the analysis can be used to isolate subsets ofbiomolecules from two or more samples with structural and/or functionalproperties that are related to differences between such biomolecules,complexes or interacting molecules that underlie the differences amongthe samples. For example, differences in specific biomolecules mayindicate protein markers of a disease and/or physiological state of aliving organism.

The subject matter of this application may involve, in some cases,interrelated products, alternative solutions to a particular problem,and/or a plurality of different uses of a single system or article.

Specifically, in one embodiment, the invention involves developing andusing methods for fractionation or separation, for example viamulti-phase partitioning, of two or more mixtures which may reflectdifferences between the mixtures related to the structural and/orfunctional characteristics of one or more molecules and/or moleculeswhich interact with such molecules. These techniques can be used toidentify markers in samples, and to techniques to using such markers fordiagnostics and other related applications.

In one aspect, the invention relates to a method for identifying one ormore tools for physiological analysis. In one embodiment, the methodinvolves determining a relative measure of interaction between prostatespecific antigen of a first mixture of species and at least first andsecond interacting components defining at least a first phase and asecond phase, respectively, of a first partitioning system. A relativemeasure of interaction is also determined between prostate specificantigen of a second mixture of species, and the first partitioningsystem. A difference is determined in the relative measure ofinteraction of the prostate specific antigen of the first mixture,versus the prostate specific antigen of the second mixture, with thefirst system. Based upon this difference, a first system is selected asa tool for determining a physiological condition of a biological systembased upon determination of a relative measure of interaction between atleast one species of a sample from the biological system and the firstsystem. Alternatively, or in addition, the at least one species of thefirst mixture and the at least one corresponding species of the secondmixture are selected as a marker for determining a physiologicalcondition of a biological system.

In another aspect, the invention involves determining a physiologicalcondition of a biological system. In one embodiment, a method for doingso involves determining a relative measure of interaction betweenprostate specific antigen arising from a sample from a biologicalsystem, and at least first and second interacting components defining atleast a first phase and a second phase, respectively, of a firstpartitioning system. From the process of determining the relativemeasure of interaction between the first species and the first andsecond interacting components of the first partitioning system, thephysiological condition of the biological system can be determined.

In another embodiment, the method involves determining a physiologicalcondition of a biological system by determining a difference between atleast a first marker of a sample from the biological system and acorresponding marker representative of a reference condition of thebiological system, without knowledge of the chemical or biologicalidentity of the first marker.

In another embodiment, a method involves determining a physiologicalcondition of a biological system by determining a difference and/orsimilarity between a first property and/or value of a propertyassociated with a marker obtained from the biological system and fromthe same marker from at least one sample with at least one referencecondition, where the marker was determined by determining a relativemeasure of interaction between at least one species of a first mixtureof species and at least first and second interacting components definingat least a first phase and a second phase, respectively, of a firstpartitioning system, determining a relative measure of interactionbetween at least one species of a second mixture of species,corresponding to the first species, and the first system, and definingthe at least one species of the first mixture of species and the atleast one species of the second mixture of species corresponding to thefirst species as the marker by denoting a difference between therelative measures of interaction of each of the species with the firstpartitioning system.

Other advantages and novel features of the invention will becomeapparent from the following detailed description of various non-limitingembodiments of the invention when considered in conjunction with theaccompanying figures. In cases where the present specification and adocument incorporated by reference include conflicting disclosure, thepresent specification shall control.

BRIEF DESCRIPTION OF THE DRAWINGS

Non-limiting embodiments of the present invention will be described byway of example with reference to the accompanying figures, which areschematic and are not intended to be drawn to scale. In the figures,each identical or nearly identical component illustrated is typicallyrepresented by a single numeral. For the purposes of clarity, not everycomponent is labeled in every figure, nor is every component of eachembodiment of the invention shown where illustration is not necessary toallow those of ordinary skill in the art to understand the invention. Inthe figures:

FIG. 1 is a RP-HPLC chromatogram of certain proteins, from a proteinmixture, having a 3.5<pI<3.9;

FIG. 2 is a RP-HPLC chromatogram of certain proteins, from the sameprotein mixture as FIG. 1, having a 4.3<pI<4.6;

FIG. 3 is a schematic block diagram of a controller according to oneembodiment of the present invention;

FIG. 4. PSA concentrations in the top and bottom phases for both normaland cancer samples according to one embodiment of the invention, asdiscussed in Example 4; and

FIG. 5 is the Receiver-Operating Characteristics (ROC) curvecorresponding to clinical data described in Example 8, in anotherembodiment of the invention.

DETAILED DESCRIPTION

The following documents are incorporated herein by reference in theirentirety: U.S. Pat. No. 6,136,960, issued Oct. 24, 2000, entitled“Method for Evaluation of the Ratio of Amounts of Biomolecules or TheirSub-populations in a Mixture,” by Chait et al.; U.S. patent applicationSer. No. 10/293,959, filed Nov. 12, 2002, entitled “Characterization ofMolecules,” by A. Chait, et al.; U.S. Patent Application Ser. No.60/478,645, filed Jun. 13, 2003, entitled “Systems and Methods forCharacterization of Molecules,” by A. Chait, et al.; U.S. PatentApplication Ser. No. 60/561,945, filed Apr. 14, 2004, entitled “Systemsand Methods for Characterization of Molecules” by Chait, et al; U.S.patent application Ser. No. 10/560,373, filed Dec. 12, 2005, entitled“Systems and Methods for Characterization of Molecules,” by Chait, etal.; and International Patent Application No. PCT/US04/019343, filedJun. 14, 2004, entitled “Systems and Methods for Characterization ofMolecules,” by Chait, et al., published as WO 2004/111655 on Dec. 23,2004.

As used in the specification and the appended claims, the singular forms“a,” “an” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a biomolecule” caninclude mixtures of a biomolecule, and the like.

Ranges may be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, another embodiment includes from the one particular valueand/or to the other particular value. Similarly, when values areexpressed as approximations, by use of the antecedent “about,” it willbe understood that the particular value forms another embodiment. Itwill be further understood that the endpoints of each of the ranges aresignificant both in relation to the other endpoint, and independently ofthe other endpoint.

As used herein, “or” is understood to mean inclusively or, i.e., theinclusion of at least one, but including more than one, of a number orlist of elements. Only terms clearly indicated to the contrary, such as“exclusively or” or “exactly one of,” will refer to the inclusion ofexactly one element of a number or list of elements.

“Optional” or “optionally” means that the subsequently described eventor circumstance may or may not occur, and that the description includesinstances where said event or circumstance occurs and instances where itdoes not.

“Analyte,” “analyte molecule,” or “analyte species” refers to amolecule, typically a macromolecule, such as a polynucleotide orpolypeptide, whose presence, amount, and/or identity are to bedetermined.

“Antibody,” as used herein, means a polyclonal or monoclonal antibody.Further, the term “antibody” means intact immunoglobulin molecules,chimeric immunoglobulin molecules, or Fab or F(ab′)₂ fragments. Suchantibodies and antibody fragments can be produced by techniques wellknown in the art, which include, for example, those described in Harlowand Lane (Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y. (1989)), Kohler et al. (Nature 256:495-97 (1975)), and U.S. Pat. Nos. 5,545,806, 5,569,825 and 5,625,126,each incorporated herein by reference. Correspondingly, antibodies, asdefined herein, also include single chain antibodies (ScFv), which maycomprise linked V_(H) and V_(L) domains and which may retain theconformation and the specific binding activity of the native idiotype ofthe antibody. Such single chain antibodies are well known in the art andcan be produced by standard methods. See, e.g., Alvarez et al., Hum.Gene Ther. 8: 229-242 (1997)). The antibodies of the present inventioncan be of any isotype, for example, IgG, IgA, IgD, IgE and IgM.

“Aqueous,” as used herein, refers to the characteristic properties of asolvent/solute system wherein the solvating substance has apredominantly hydrophilic character. Examples of aqueous solvent/solutesystems include those where water, or compositions containing water, arethe predominant solvent.

“Partitioning system,” as used herein, refers to any material having atleast two phases, sections, areas, components, or the like, at least twoof which can interact differently with at least one species to whichthey are exposed. For example, a partitioning system can includedifferent areas of a solid surface, which can interact differently witha particular molecule exposed to the different sections, a multi-phasesystem such as a multi-phase liquid system, e.g., an aqueous/non-aqueoussystem or an aqueous multi-phase system (defined below) to which one ormore species can be exposed and optionally dissolved, at least some ofwhich species can interact differently with different phases. Forexample, a particular species may have a greater affinity for one phaserather than another phase to the extent that a multi-phase partitioningsystem can isolate a species from a mixture, or cause a species topartition at least in some way differently between the phases.

“Aqueous multi-phase system,” as used herein, refers to an aqueoussystem which includes greater than one aqueous phase in which an analytespecies can reside, and which can be used to characterize the structuralstate of the analyte species according to the methods described herein.For example, an aqueous multi-phase system can separate at equilibriuminto two, three, or more immiscible phases. Aqueous multi-phase systemsare known in the art and this phrase, as used herein, is not meant to beinconsistent with accepted meaning in the art. Examples of variousaqueous multi-phase systems, and their compositions, are described morefully below.

An “interacting component” means a component, such as a phase ofmulti-phase system, that can interact with a species and provideinformation about that species (for example, an affinity for thespecies). Multiple interacting components, exposed to a species, candefine a system that can provide a “relative measure of interaction”between each component and the species. An interacting component can beaqueous or non-aqueous, can be polymeric, organic (e.g. a protein, smallmolecule, etc.), inorganic (e.g. a salt), or the like, or anycombination thereof. A set of interacting components can form a systemuseful in and in part defining any experimental method which is used tocharacterize the structural state of a species such as an analytespecies according to the methods described herein. Typically, a systemof interacting components can measure the relative interaction betweenthe species and at least two interacting components. An aqueousmulti-phase system is an example of a system of interacting components,and it is to be understood that where “aqueous system” or “aqueousmulti-phase system” is used herein, this is by way of example only, andany suitable system of interacting components can be used.

Where aqueous two-phase and aqueous multi-phase systems are describedherein, it is to be understood that other systems, as used herein,systems analogous to those comprising only aqueous solutions orsuspensions can be used. For example, an aqueous two-phase system caninclude non-aqueous components in one or more phases that are not liquidin character. In this aspect, multi-phase systems also refers to relatedtechniques that rely on differential affinity of the biomolecule to onemedia versus another, wherein the transport of the biomolecule betweenone medium and, optionally, another medium occurs in an aqueousenvironment. Examples of such multi-phase systems include, but are notlimited to, HPLC columns or systems for liquid-liquid partitionchromatography, as are known to those of ordinary skill in the art.

“Relative measure of interaction,” with reference to a particularspecies as used herein, means the degree to which the species interactswith another species or with a phase of a multi-phase system in arelative sense. For example, a particular species may have a greateraffinity for one phase of a multi-phase system rather than another phaseor phases, the degree to which it interacts with or resides in, thatphase as opposed to other phases defines its relative measure ofinteraction. Relative measures of interaction, in the context of thepresent invention, are generally determined in a ratiometric manner,rather than an absolute manner. That is, where a species can interactwith each phase of a two-phase system but resides more preferably in onethan the other, the present invention typically makes use of informationas to the ratio of concentration of the species in each of the twophases, but not necessarily of the absolute concentration of the speciesin either phase. In other cases, the interaction can be an interactionbased not upon residence of a particular species within a particularsolvent or fluid carrier, but interaction with a solid surface such as asolid phase of a chromatography column where the relative measuremanifests itself in elution time, or can involve geometric or spatialinteraction such as a particular species interaction with a poroussubstrate as opposed to that of a different species or a differentsubstrate.

“Partition coefficient,” as used herein, refers to the coefficient whichis defined by the ratio of chemical activity or the concentrations of aspecies in two or more phases of a multi-phase system at equilibrium.For example, the partition coefficient (K) of an analyte in a two-phasesystem can be defined as the ratio of the concentration of analyte inthe first phase to that in the second phase. For multi-phase systems,there can be multiple partition coefficients, where each partitioncoefficient defines the ratio of species in first selected phase and asecond selected phase. It will be recognized that the total number ofpartition coefficients in any multi-phase system will be equal to thetotal number of phases minus one.

For heterogeneous phase systems, an “apparent partition coefficient,” asused herein, refers to a coefficient which describes informationobtained from alternative techniques that is correlated to the relativepartitioning between phases. For example, if the heterogeneous two-phasesystem used is an HPLC column, this “apparent partition coefficient” canbe the relative retention time for the analyte. It will be recognized bythose of ordinary skill in the art that the retention time of ananalyte, in such a case, reflects the average partitioning of theanalyte between a first, mobile phase and a second, immobile phase.Also, it will be recognized that other, similarly determinableproperties of analytes can also be used to quantify differences inphysical properties of the analytes (e.g. in other techniques) and are,therefore, suitable for use as apparent partition coefficients.

“Bind,” as used herein, means the well-understood receptor/ligandbinding, as well as other nonrandom association between a biomoleculeand its binding partner. “Specifically bind,” as used herein, describesa binding partner or other ligand that does not cross reactsubstantially with any biomolecule other than the biomolecule orbiomolecules specified. Generally, molecules which preferentially bindto each other are referred to as a “specific binding pair.” Such pairsinclude, but are not limited to, an antibody and its antigen, a lectinand a carbohydrate which it binds, an enzyme and its substrate, and ahormone and its cellular receptor. As generally used, the terms“receptor” and “ligand” are used to identify a pair of bindingmolecules. Usually, the term “receptor” is assigned to a member of aspecific binding pair, which is of a class of molecules known for itsbinding activity, e.g., antibodies. The term “receptor” is alsopreferentially conferred on the member of a pair that is larger in size,e.g., on lectin in the case of the lectin-carbohydrate pair. However, itwill be recognized by those of skill in the art that the identificationof receptor and ligand is somewhat arbitrary, and the term “ligand” maybe used to refer to a molecule which others would call a “receptor.” Theterm “anti-ligand” is sometimes used in place of “receptor.”

“Molecule-molecule interaction”, such as biomolecule-biomoleculeinteraction, protein-protein interaction, and the like means aninteraction that typically is weaker than “binding,” i.e., aninteraction based upon hydrogen bonding, van der Waals binding, Londonforces, and/or other non-covalent interactions that contribute to anaffinity of one molecule for another molecule, which affinity can beassisted by structural features such as the ability of one molecule toconform to another molecule or a section of another molecule.Molecule-molecule interactions can involve binding, but need not.

“Biomolecule,” as used herein, means a molecule typically derived froman organism, and which typically includes building blocks includingnucleotides, and the like. Examples include, but are not limited to,peptides, polypeptides, proteins, protein complexes, nucleotides,oligonucleotides, polynucleotides, nucleic acid complexes, saccharides,oligosaccharides, carbohydrates, lipids, etc., as well as combinations,enantiomers, homologs, analogs, derivatives and/or mimetics thereof.

“Species,” as used herein, refers to a molecule or collection ofmolecules, for example, an inorganic chemical, an organic chemical, abiomolecule, or the like. In the present invention, species generallyare biomolecules.

“Corresponding species,” as used herein, means at least two differentspecies that are identical chemically or, if they differ chemicallyand/or by molecular weight, differ only slightly. Examples ofcorresponding species include structural isoforms of proteins, proteinsor other molecules that are essentially identical but that differ inbinding affinity with respect to another species or plural species, havedifferent higher-order structure, e.g., differing in secondary ortertiary structure but not differing or not differing significantly inchemical sequence. In general, corresponding species are species thatmay be arranged differently (isoforms, isomers, etc.) but are composedof the same or essentially the same chemical building blocks.

“Detectable,” as used herein, refers the ability of a species and/or aproperty of the species to be discerned. One example method of renderinga species detectable is to provide further species that bind or interactwith the first species, where the species comprise(s) a detectablelabel. Examples of detectable labels include, but are not limited to,nucleic acid labels, chemically reactive labels, fluorescence labels,enzymic labels and radioactive labels.

“Mimetic,” as used herein, includes a chemical compound, an organicmolecule, or any other mimetic, the structure of which is based on, orderived from, a binding region of an antibody or antigen. For example,one can model predicted chemical structures to mimic the structure of abinding region, such as a binding loop of a peptide. Such modeling canbe performed using standard methods (see, for example, Zhao et al., Nat.Struct. Biol. 2: 1131-1137 (1995)). The mimetics identified by methodssuch as this can be further characterized as having the same bindingfunction as the originally identified molecule of interest, according tothe binding assays described herein.

Alternatively, mimetics can also be selected from combinatorial chemicallibraries in much the same way that peptides are. See, for example,Ostresh et al., Proc. Natl. Acad. Sci. U.S.A. 91: 11138-11142 (1994);Dorner et al., Bioorg. Med. Chem. 4: 709-715 (1996); Eichler et al.,Med. Res. Rev. 15: 481-96 (1995); Blondelle et al., Biochem. J. 313:141-147 (1996); Perez-Paya et al., J. Biol. Chem. 271: 4120-6 (1996).

“Solid support,” as used herein, means the well understood solidmaterial to which various components of the invention are physicallyattached, thereby immobilizing the components of the present invention.The term “solid support,” as used herein, means a non-liquid substance.A solid support can be, but is not limited to, a membrane, sheet, gel,glass, plastic or metal. Immobilized components of the invention may beassociated with a solid support by covalent bonds and/or vianon-covalent attractive forces such as hydrogen bond interactions,hydrophobic attractive forces and ionic forces, for example.

“Structure,” “structural state,” “configuration” or “conformation,” asused herein, all refer to the commonly understood meanings of therespective terms, for example, as they apply to biomolecules such asproteins and nucleic acids, as well as pharmacologically active smallmolecules. In different contexts, the meaning of these terms will vary,as is appreciated by those of skill in the art. The structure orstructural state of a molecule refers generally not to the buildingblocks that define the molecule but the spatial arrangement of thesebuilding blocks. The configuration or confirmation typically definesthis arrangement. For instance, the use of the terms primary, secondary,tertiary or quaternary, in reference to protein structure, have acceptedmeanings within the art, which differ in some respects from theirmeaning when used in reference to nucleic acid structure (see, e.g.,Cantor and Schimmel, Biophysical Chemistry, Parts I-III). Unlessotherwise specified, the meanings of these terms will be those generallyaccepted by those of skill in the art.

“Physiological conditions,” as used herein, means the physical,chemical, or biophysical state of an organism. As most typically used inthe context of the present invention, physiological condition refers toa normal (e.g., healthy in the context of a human) or abnormal (e.g., ina diseased state in the context of a human) condition.

“Marker,” as used herein, is a species that can be a carrier ofinformation regarding a physiological state of a biological environmentwithin which it resides. A marker can exhibit at least two differentproperties or values of a specific property or properties (e.g.,structural conformation, binding affinity for another species, etc. butnot solely different amounts of the species) that correspond to and/orthat represent information regarding the two or more physiologicalstates of environments within which they reside. For example, a markermay be a protein that is structurally modified between a first staterepresentative of a healthy system within which it resides and a secondstructural state (different conformation) representative of a diseasesystem within which it resides.

The state of a molecule, such as a biomolecule, at whatever level ofdetail, can be affected by many different factors including, but notlimited to, changes in the chemical structure of the molecule (e.g.,addition, deletion or substitution of amino acids in proteins, covalentmodification by chemical agents or cleavage by chemical or thermaldegradation, addition or deletion of carbohydrates to the structure,etc.), interactions with one or more other biomolecules or ligands, andthe like. Evaluation of different states can be used as one method ofdetermining the potential effectiveness of different molecules,condition of the molecules, condition or state of an environment (e.g.,a mixture of species) within which the molecule resides, and the like.

The present invention involves the investigation of the state ofmolecules. The invention is described in the context of studiesinvolving biomolecules and/or molecules able to interact withbiomolecules, but the invention can apply to essentially any molecularspecies and/or interaction, whether biological, biochemical, chemical,or other species, and those of ordinary skill in the art will understandhow the invention can be used in the context of non-biologicalmolecules. It is to be understood that whenever “biomolecules” is usedin the description of the invention, any non-biological molecule alsocan be used or studied.

In one aspect, the present, in some embodiments, invention involvestechniques for determining information about the composition of amixture of biomolecules and/or molecules which interact withbiomolecules. The mixture may originate from biological material, suchas human clinical sample or other biological fluid, tissue, cells, asubject, etc., or the mixture, may be a synthetic mixture. The mixturecan come from a biological system which, as used herein, means a humanor non-human mammal, including, but not limited to, a dog, cat, horse,cow, pig, sheep, goat, chicken, primate, rat, and mouse, or a bacteria,virus, fungus, or of plant origin.

The invention also relates to developing and determining characteristics(quantitative and/or qualitative) of a mixture that are obtained, forexample, via processing using multi-phase partitioning, which canreflect certain structural and functional characteristics ofbiomolecules or molecules that interact with biomolecules in theoriginal mixture. These characteristics can be used, for example, forestablishing relationships between the composition of the mixture andthe physiological state of the biological source of the mixture e.g.,the state of health or disease of a subject. These characteristics canalso be used to design experimental conditions for subsequentfractionation of the mixtures into subsets enriched in the molecule(s)of interest for the purpose of the analysis, while simultaneouslyreduced in the total number of different molecule(s) in some cases. Thesystems and methods of the present invention can also be useful fordetecting, classifying, and/or predicting changes in a mixture ofbiomolecules or molecules that interact with biomolecules. For example,the mixture may be a synthetic mixture, or a mixture associated with aparticular disease or physiological state of a living organism, cells,tissues, or biological liquids. The systems and methods of the presentinvention can also be used to detect changes to a predetermined set ofbiomolecules in a biological mixture and these changes could further beused to detect and classify a diagnostic that is related to suchchanges.

Examples of such changes in a mixture can be the differences in aproperty of a species of the mixture, such as its conformation,structure and/or interaction tendency with respect to another moleculeor molecules (e.g., its binding affinity or other interactioncharacteristic with respect to another molecule or molecules). Forexample, if the mixture includes proteins or other biomolecules, suchchanges may be induced through primary sequence modification, bydegradation of the proteins or other biomolecules through chemical,thermal, or other degradation mechanisms, by interaction with othermolecules and/or biomolecules, by interaction with low molecular weightcompounds (e.g., hormones, peptides, vitamins, cofactors, etc.), bychanges in the relative content or concentration of the constituents ofthe mixture, by reactions such as enzymatic reactions, etc. The systemsand methods of the present invention can be used, in some cases, todetect, analyze and/or characterize biological materials, including butnot limited to, polypeptides, proteins, carbohydrates, nucleic acids,polynucleotides, lipids, sterols, and mixtures or derivatives thereof,e.g., for the purpose of detection of, or onset of, a particular diseaseor physiological state, monitoring its progress, treatment, etc.

Comparison and classification steps involved in the invention can makeuse of additional information not necessarily related to (not directlyderived from) the analytical methods of the invention. For example,blood pressure, temperature, blood glucose level, and/or essentially anyother measurable physiological condition can be used in conjunction withtechniques of the invention to analyze one or more physiologicalconditions.

As mentioned, multiple partitioning steps can take place so thatadditional information and/or sensitivity can be obtained. For example,prior to determining relative measures of interaction of each species ineach of two or more different mixtures, and following partitioning ofboth mixtures in two or more partitioning systems of identical (ornearly identical) composition, a quantity of the first and/or the secondinteracting components of both systems containing the mixtures can befurther introduced into a second set of two identical systems with atleast two interacting components. Then, partitioning of both second setsof systems for both mixtures takes place, and relative measures ofinteraction for each species in each mixture can be determined. Thespecies that differ among the mixtures by differences in their relativemeasures of interaction can be denoted. Then, optionally, a set of oneor more species that partition differently can be further selected as aset of markers that are subsequently used to classify additionalmixtures as similar to either the first or second mixtures. Determiningsteps can be performed separately for each species in connection withthe set of markers, or simultaneously. Species-specific probes can beused, such as antibodies. Comparison or classification steps todetermine markers and use such markers can involve the use ofmathematical or statistical techniques known to those of ordinary skillin the art.

It will be recognized by those of ordinary skill in the art that thesebiological materials can be found in any suitable form, for example, inthe form of extracts from natural sources, biological liquids,collections of molecules generated by combinatorial chemical orbiochemical techniques and combinations thereof, synthetically created,etc.

In one embodiment, the present invention provides a method to determinecertain conditions under which variations among samples representingdifferent compositions (or mixtures of species) could be detected, i.e.,determining a set of criteria and/or system components as a “tool,” or apart of a tool, to determine information. For example, the ability of asystem to determine a relative measure of interaction between a speciesand one or more interacting components that can define one or morephases of the system can serve as an important tool or component of sucha tool. Specifically, as one example, the partitioning of theconstituents of a sample between two phases having different chemical orbiochemical affinities or other characteristics, such as solventstructures, may separate the constituents by their relative affinity formedia of different properties or composition. This separation techniquethus can include or, alternatively, can be unlike those typically usedin proteomics or similar techniques, e.g., 2-D gel electrophoresis, inwhich charge and size differences are the two dimensions used toseparate the constituents of a sample. In some cases, e.g., for manyapplications in proteomics, the present invention provides the abilityfor performing sequential or serial partitioning, with either the sameof different conditions, which may result in additional amplification ofdifferences in the fractionated samples. These fractions may be furtheranalyzed using standard proteomics techniques.

As mentioned elsewhere herein, aqueous multi-phase (e.g., two-phase)partitioning systems are well-suited for use in many or most embodimentsof the invention, but other partitioning systems can be used. Where“aqueous two-phase partitioning” or “aqueous multi-phase partitioning”is used, it is to be understood that other systems can be used.Partitioning of a biopolymer in aqueous two-phase systems may depend onits three-dimensional structure, type and topography of chemical groupsexposed to the solvent, etc. Changes in the 3-D structure of a receptorinduced by some effect, e.g., by binding of a ligand binding or bystructural degradation, also can change the topography of solventaccessible chemical groups in the biomolecule, or both the topographyand the type of the groups accessible to solvent. One result of thesechanges may be an alteration in the partition behavior of thebiomolecule and/or the ligand-bound receptor.

In some cases, the level of concentration of biomolecules in biologicalsamples is strongly dependent upon genotyping. Thus, identification ofdifferences in biomolecules attributable to diseased verses normalstates may necessitate using a statistically significant number ofsamples to negate the effect of natural genetic variations in manycases. In the present invention, in many cases, the effect of geneticvariability leading to under- or overexpression can be separated fromdifferences to biomolecules that are traced to their diseased versusnormal states. This separation can be achieved by subjecting a sample orother mixture of species containing biomolecules or other molecules topartitioning in one or more different systems, and determining arelative measure of interaction between at least one species in thesample/mixture with various components of the system(s). This can bedone, for example, by separating, using conventional techniques, the twointeracting components of each sample, calculating the partitioncoefficient for each species in the diseased and normal samples, andselecting the species exhibiting different partition coefficients forfurther analysis and identification. As specific examples, the relativemeasure of interaction can involve fractionating at least a portion ofthe first portion and second portion (and/or more portions) of thesystem. This fractionating can involve electrophoresis such asone-dimensional electrophoresis, two-dimensional electrophoresis, caninvolve liquid or other chromatography, can involve performing massspectrometry on at least a portion of the first, second (and,alternately, more portions) of the system, etc. Different partitioncoefficients typically are not related to the absolute level ofexpression of each species, but instead, may be related to changes tothe structure, binding to other molecules or other changes of relevanceto their biological effects, etc. Thus, the present invention provides,in one set of embodiments, means for the identification of changes tobiomolecules in a biological mixture inherent to their function and nottheir absolute level, without necessarily requiring a large statisticalnumber of samples to negate the effect of individual variability in theexpression levels.

Once the biomolecules of interest are identified using the abovetechnique, a subset can be selected providing acceptablesensitivity/specificity diagnostics levels for an underlyingphysiological condition, e.g., a disease. Rapid and specificquantification techniques are readily available to those of ordinaryskill in the art which can be used to quantify the concentration of eachof the biomolecules in the subset using standard methods and techniquesdirectly in the biological sample, e.g., using antibodies in an EnzymeLinked ImmunoSorbent Assay (ELISA). The concentrations in the twointeracting components of each system can be used to calculate thevalues of the partition coefficients. Changes to the individual valuesof the partition coefficients thus may indicate certain changes to thebiomolecules. In some cases, the change to the partition coefficient ofa single biomolecule can result in a definitive diagnostics. In othercases, the use of a pattern of partition coefficient values (a“signature”) can be used to enhance the specificity of the method. Inyet other cases, partitioning of the samples in multiple systems andperforming the steps above, then observing the pattern of values for oneor more biomolecules, can provide an alternative way to constructing asensitive and specific diagnostics method.

Similarly, such changes may be detected using other systems and methodswhich have an underlying dependence upon the topography and/or the typesof solvent accessible groups. Examples of such other methods include,but are not limited to, column liquid-liquid partition chromatography(LLPC), a heterogeneous two-phase system, or a multiphase heterogeneoussystem. In some cases, an apparent partition coefficient may begenerated that expresses the relative changes in the averagepartitioning between a first and a second phase. For example, in LLPC,the retention volume of a receptor may be used as the apparent partitioncoefficient.

Aqueous two-phase systems are well-known to those of ordinary skill inthe art, and can arise in aqueous mixtures of different water-solublepolymers or a single polymer and a specific salt. When two or morecertain polymers, e.g., dextran (“Dex”) and polyethylene glycol (“PEG”),or one or more certain polymers and one or more inorganic salts, e.g.polyvinylpyrrolidone (“PVP”) and sodium sulfate, are mixed in waterabove certain concentrations, the mixture can separate into two (ormore) immiscible aqueous phases under certain conditions. There is adiscrete interfacial boundary separating any two phases, for example,such that one is rich in one polymer and the other phase is rich in theother polymer or the inorganic salt. The aqueous solvent in one or bothphases may provide a medium suitable for biological products. Two-phasesystems can also be generalized to multiple phase system by usingdifferent chemical components, and aqueous systems with a dozen or morephases are known in the art and can be used in connection with theinvention.

When a species is introduced into such a two-phase system, it maydistribute between the two phases. In this and other systems, thespecies can be found at different concentrations within each phase, orcan be at the same concentration within each phase. Partitioning of asolute can be characterized by the partition coefficient “K,” defined asthe ratio between the concentrations of the solute the two immisciblephases at equilibrium. It has previously been shown that phaseseparation in aqueous polymer systems may result from different effectsof two polymers (or a single polymer and a salt) on the water structure(B. Zavlavsky, Aqueous Two-Phase Partitioning: Physical Chemistry andBioanalytical Applications, Marcel Dekker, New York, 1995). As theresult of the different effects on water structure, the solvent featuresof aqueous media in the coexisting phases can differ from one another.The difference between phases may be demonstrated by techniques such asdielectric, solvatochromic, potentiometric, and/or partitionmeasurements.

The basic rules of solute partitioning in aqueous two-phase systems havebeen shown to be similar to those in water-organic solvent systems(which can also be used as systems in the present invention). However,what differences do exist in the properties of the two phases in aqueouspolymer systems are often very small, relative to those observed inwater-organic solvent systems, as would be expected for a pair ofsolvents of the same (aqueous) nature. The small differences between thesolvent features of the phases in aqueous two-phase or multi-phasesystems can be modified so as to amplify the observed partitioning thatresults when certain structural features are present.

It is known that the polymer and salt compositions of each of the phasesusually depend upon the total polymer and/or salt composition of anaqueous two-phase system. The polymer and/or salt composition of a givenphase, in turn, usually governs the solvent features of the aqueousmedia in this phase. These features include, but are not limited to,dielectric properties, solvent polarity, ability of the solvent toparticipate in hydrophobic hydration interactions with a solute, abilityof the solvent to participate in electrostatic interactions with asolute, and hydrogen bond acidity and basicity of the solvent. All theseand other solvent features of aqueous media in the coexisting phases maybe manipulated by selection of polymer and salt composition of anaqueous two-phase system. These solvent features of the media may governthe sensitivity of a given aqueous two-phase system toward a particulartype of solvent accessible chemical groups in the receptor. Thissensitivity, type, and topography of the solvent accessible groups intwo different proteins, for example, can determine the possibility ofseparating proteins in a given aqueous two-phase system.

In some cases, a particularly sensitive system may be required, i.e., asystem that is very sensitive to, and able to determine relativemeasures of interaction with respect to, two very similar species. Thissensitivity may be of importance when, for example, subtle differencesare being detected between the conformational changes in a receptorinduced by binding of closely related chemical compounds. The presentinvention provides efficient and successful systems and methods forscreening aqueous phase compositions to identify and/or amplifydifferences between the compositions of two mixtures. By utilizing awide variety of different conditions to screen each molecule, asdescribed herein, different partitioning behavior may be obtainedreliably without the need to fully understand the underlying theory ofaqueous two-phase partitioning, or any of the other related orsubstitutable techniques.

Biomolecules such as proteins, nucleic acids, etc. may be distributedbetween the two or more phases when placed into such a system. Forexample, in the case where phase-forming polymers are used, solutionscomprising one or more of the two polymers and the biomolecule may bemixed together such that both phase-forming polymers and the biomoleculeare mixed. The resulting solution is resolved and a two-phase system isformed. Optionally, centrifugation can be used to enhance separation ofthe phases. It will be recognized by those of ordinary skill in the artthat partitioning behavior of a biomolecule may be influenced by manyvariables, such as the pH, the polymers used, the salts used, factorsrelating to the composition of the system, as well as other factors suchas temperature, volume, etc. Optimization of these factors for desiredeffects can be accomplished by routine practice by those of ordinaryskill in the relevant arts, in combination with the current disclosure.

Evaluation of data from partitioning of a biomolecule can involve use ofthe partition coefficient, in some embodiments of the invention. Forexample, the partition coefficient of a protein can be taken as theratio of the protein in first phase to that in the second phase in abiphasic system. When multiple phase systems are formed, there can bemultiple independent partition coefficients, each of which can bedefined between any two phases. It will be recognized that the partitioncoefficient for a given biomolecule of a given conformation will be aconstant if the conditions and the composition of the two-phase systemto which it is subjected remain constant. Thus, if changes are observedin the partition coefficient for a protein upon addition of a potentialbinding partner, these changes can be presumed to result from changes inthe protein structure caused by formation of a protein-binding partnercomplex. The partition coefficient K, as used herein, is a specificallymathematically defined quantity as further described below, and the termincludes coefficients representing the relative measure of interactionbetween a species and at least two interacting components. It shouldalso be recognized that differences between partition coefficients ofcorresponding species in two or more mixtures could indicate, inaddition to potential structural changes, also binding or lack ofbinding of such species to other species in the mixtures.

In a non-limiting example of one partitioning system, aqueous multiphasesystems are known to be formable from a variety of substances. Forexample, in order to determine the partition coefficient of a protein(or a mixture of a protein with another compound) to be analyzed,concentrated stock solutions of all the components (polymer 1, e.g.,dextran; polymer 2, e.g., PEG, polyvinylpyrrolidone, salts, etc.) inwater can be prepared separately. The stock solutions of phase polymers,salts, and the protein mixture can be mixed in the amounts andconditions (e.g., pH from about 3.0 to about 9.0, temperature from about4° C. to 60° C., salt concentration from 0.001 to 5 mol/kg) appropriateto bring the system to the desired composition and vigorously shaken.The system can then be allowed to equilibrate (resolve the phases).Equilibration can be accomplished by allowing the solution to remainundisturbed, or it can be accelerated by centrifugation, e.g., for 2-30minutes at about 1000 g to 4000 g, or higher. Aliquots of each settled(resolved) phase can be withdrawn from the upper and/or lower phases (orfrom one or more phases, if multiple phases are present). Theconcentration of molecule(s) can be determined for each phase.

Different assay methods may be used to determine the relative measuresof interaction between species and interacting components, e.g. in theform of the concentration of the biomolecules in each phase of amulti-phase system. The assays will often depend upon the identity andtype of biomolecule present. Examples of suitable assay techniquesinclude, but are not limited to, spectroscopic, immunochemical,chemical, fluorescent, radiological and enzymatic assays. When thebiomolecule is a peptide or protein, the common peptide or proteindetection techniques can be used. These include, but are not limited to,direct spectrophotometry (e.g., monitoring the absorbance at 280nanometers) and dye binding reactions with Coomassie Blue G-250 orfluorescamine, o-phthaldialdehyde, or other dyes and/or reagents.Alternatively, if the protein is either an antibody or an antigen,certain immunochemical assays can be used in some cases.

The concentration of the biomolecule(s) in each phase can be used todetermine the partition coefficient of the sample under the particularsystem conditions. Since the partition coefficient reflects the ratio ofthe two concentrations, the absolute values are not typically required.It will be recognized that this can allow certain analytical proceduresto be simplified, e.g., calibration can be eliminated in some instances.It also may have significant advantage for negating the effect ofnatural variability in the absolute concentration of proteins in samplesobtained from, e.g., biological systems, when comparing two or moresamples, thus focusing on those changes detected as differences in thepartition coefficient relevant to changes to the structure of theindividual species in the samples.

It should be recognized by those skilled in the art that the steps inabove description of obtaining the partition coefficient could besubstituted by other steps or measurements. Depending on the size,volumes, amount of the biomolecule, detection system, discrete orcontinuous operation using either liquid-liquid or liquid-solidportioning, other processes that effectively result in results describedherein could be developed. Such modifications and different processesshould not limit the scope of this complete invention.

The partition coefficient can then be compared with other partitioncoefficients. For example, a partition coefficient for a species can becompared to the partition coefficients for the species under differentconditions, a partition coefficient for a species can be compared to thepartition coefficients for the species when combined with other species,a set of partition coefficients for a species can be compared to othersets of partition coefficients, etc. This comparative information can beobtained at the same time or near the same time and in the same systemor a similar system as is used to determine the interactioncharacteristics of the molecules of interest, or can be provided aspre-prepared data in the form of charts, tables, or electronicallystored information (available on the internet, disc, etc.)

In one embodiment of the present invention, proteins or otherbiomolecular mixtures from an experimental sample and from a referencesample (determined simultaneously, previously, or subsequently, asdescribed above) may be caused to partition in a variety of differentaqueous two-phase systems, e.g. formed by different types of polymers,such as Dextran and PEG or Dextran and Ficoll, by the same types ofpolymers with different molecular weights, such as Dextran-70 andPEG-600 or Dextran-70 and PEG-8,000, by the same polymers but containingdifferent in type and/or concentration salt additives, different buffersof different pH and concentration, etc. The overall partitioncoefficients for the mixtures determined using a particular assayprocedure (e.g., same for both samples) can be determined in all of thesystems. In one embodiment, the systems displaying different partitioncoefficients for the mixtures under comparison may be selected as aseparation medium, for example, for further fractionation and/orcharacterization of the mixtures. In another embodiment, mixtures arepartitioned using one or more standard systems with known properties,e.g., those providing enhanced sensitivity levels towards hydrophobic orionic interactions. In such a case, the individual partitioncoefficients of the species comprising the mixtures may be determinedfollowing separation of the mixtures in the phases and/or comparedbetween two or more mixtures.

The reasons for the observed differences in the partition behavior ofthe two samples do not have to be scientifically characterized for suchdifferences to be useful for many applications, e.g., for diagnostics.Such differences, resulting in partitioning behavior, may arise due tomultiple reasons, including relative compositional, structural, orconformational differences in the samples when exposed to aqueous mediaof different solvent structures.

In one set of embodiments, the systems and methods proposed hereinprovide techniques for the separation and fractionation of proteinswhile preserving complexes and biomolecular interactions that may be ofinterest to distinguishing among samples. The solvent media in aqueouspartitioning may be selected to be compatible with the mixture ofbiomolecules. The solvent media may also be selected to preserve thehigher-order structures, as well as non-covalent binding amongbiomolecules such as proteins, small molecular weight ligands, etc. Forexample, appearance or disappearance of complexes by the methods of thisinvention can be useful for diagnostics and other applications.

One aspect of the present invention provides systems and methods able todistinguish among different samples, without being rigidly tied to fewseparation dimensions or variables, such as charge and/or size. Onenon-limiting example application of the present invention is to providean adjustable separation dimension, in which changes to individualspecies can be uncovered via determination of their individual partitioncoefficients, enabling detection and identification of changes that cannot be detected using conventional separation means, such as molecularsize or charge.

In one embodiment, the present invention can be used to discover one ofmore biomolecules in a biological sample, which is changed betweennormal and diseased state of the underlying organism. In this case, aset of typically multiple systems, each known to provide sensitivity tostructural changes leading to differences in their hydrophobic, ionic,etc. interactions with the interacting components, can be tested withthe same samples. One or more species can be identified as markers inone or more systems using techniques described herein. This marker ormarkers can subsequently be used for diagnostics applications.

In yet another embodiment, the set of markers and the associated systemsin which such markers were discovered can be used during diagnosticsscreening. In this case, the diagnostics test can include one or more ofthe following steps which can be carried out in any order suitable forsuch screening: (1) Partitioning the sample in one or more of thesystems which were used during the marker discovery study; (2) Measuringthe marker(s) concentration(s) using specific assay using, e.g.,antibodies, in each of the interacting components of the systems; (3)Calculating the partition coefficients for each individual biomarker inone or more systems; (4) Comparing the values to those representingnormal and diseased states which were obtained during the markerdiscovery study using any combination of statistical or mathematicaltechniques; and (5) Denoting a diagnostics based on such a comparison.

Without a loss of generality, as an example, searching for a biomarkeror a set of biomarkers (e.g., to increase clinical specificity) denotinga disease can involve one or more of the following steps, again, carriedout in any suitable order:

-   -   1. Prepare one or more aqueous two-phase partitioning systems.    -   2. Add samples of plasma (homogenized tissue, urine, saliva,        etc.) corresponding to normal and diseased state origins.    -   3. Partition the samples in the aqueous two-phase systems.    -   4. Remove aliquots from both phases of the aqueous two-phase        systems for each sample. After this step there will be two        aliquots for each sample.    -   5. Perform additional separation steps (e.g., 2D gel        electrophoresis or 2D HPLC) to separate the proteins in each        aliquot.    -   6. Quantitate each protein in each aliquot.    -   7. Calculate the individual partition coefficient for each        protein in each set of aliquots corresponding to the same        sample.    -   8 Compare the partition coefficients for each protein (e.g., in        the same spot on the charge/size map on a gel) for the normal        vs. diseased states.    -   9. Select one or more proteins (even without knowing their        identity) as potential biomarker by their different values of        the partition coefficients for the two types of samples.    -   10. Optionally perform additional identification steps such as        LC-MS/MS. It should be noted that discovering and selecting the        marker(s) in the present invention does not require the steps of        protein identification.

It should also be noted that the marker is comprised in the presentinvention of the species selected in the manner described above, andanother important tool in the process can involve the specificcomposition of the aqueous two-phase partitioning system or other systemthat can be used to determine relative measures of interaction. Itshould be recognized that multiple partitioning systems of differentcompositions can be used in the above procedure. The final selection ofa set of markers most useful for subsequent diagnostics dependstypically on a trade off among the competing attributes of the increasein specificity and cost when additional biomarkers are included in thefinal set.

Once a set of biomarkers is discovered using the above protocol, adiagnostics screening test can be devised, without a loss of generality,according in the following manner:

Upon a preliminary step performed once during the preparation of thescreening test, an appropriate protein-specific concentration assay isdeveloped for each of the proteins in the biomarker set (typicallyantibodies). Typically once a biomarker is determined using a particularpartitioning system, aliquots of the separated biomarker can be used tostudy its properties and sequence, and antibodies developed forsubsequent quantitation of the biomarker directly in complex mixturesusing, e.g., ELISA.

Then, the screening test is comprised of the following, again, in anysuitable order:

-   -   1. Obtain a sample of plasma (homogenized tissue, urine, saliva,        etc.) corresponding to unknown state (normal or diseased).    -   2. Add aliquots of the sample to the partitioning system used        during the discovery of the biomarkers. If more than one system        was used, repeat the same step for each different partitioning        system.    -   3. Perform partitioning of the sample in each of the systems.    -   4. Use the protein specific assay for each protein to quantitate        the concentration of each of the proteins in the biomarkers set,        in each of the two phases of each partitioning system.    -   5. Calculate the partition coefficient for each of the proteins        in the biomarkers set.    -   6. Compare, using appropriate statistical or other techniques        the partition coefficients from the sample of unknown origin to        those corresponding to the normal and diseased states.    -   7. Classify the unknown sample as diagnostically similar to one        of the known samples.

In another embodiment, a screening test can be accomplished withoutrequiring partitioning as described above. Once a biomarker isdiscovered and selected using techniques described herein, it typicallyrefers to a protein that is structurally different in the two samples(e.g., diseased vs. normal). Once the protein is isolated, identifiedusing standard techniques such as LC-MS/MS, and its sequence denoted, aseparate procedure can be invoked in some cases to design and raiseantibodies that directly differentiate between the two forms of theprotein that define the marker. These so-called multi-site antibodiescan be used in lieu of the partitioning step in the screening procedure,e.g., as follows, in any suitable order:

-   -   1. Obtain a sample of plasma (homogenized tissue, urine, saliva,        etc.) corresponding to unknown state (normal or diseased).    -   2. Directly test the sample with antibody specific to one form        of the marker.    -   3. Classify the unknown samples as diagnostically similar to one        of the known samples.

In the protocol described above, the use of form-specific antibody toreplace the use of partitioning to delineate different forms of thebiomarker may be only possible once the biomarker is discovered usingtechniques described herein.

It is also noted that the present invention carries the same benefit forthe more general and practical case in which the biomarker represents amixture of forms of the same protein. In such a case, changes in thedistribution or relative amounts of the different forms of the sameprotein will result in a different partitioning behavior of thebiomarker, which will be detected using techniques described herein.Therefore the use of the term “biomarker” in the present invention maydenote either a single molecule that with different forms between thetwo samples, or mixtures of two or more forms of the same molecule thatdiffer in the relative amounts of the forms between the two samples.

In connection with all aspects of the invention, a variety of studiescan take place, both at the level of determining tools for physiologicalanalysis and carrying out physiological analysis itself. For example,tools for determining analysis procedures can involve taking samplesfrom a single individual or multiple individuals. In one embodiment, apositive sample and a control sample can be taken from a singleindividual. For example, an individual may have a tumor and a positivesample may be a portion of the tumor, where a control sample is from anon-tumorous portion of the individual. The samples, both positive andcontrol, can be taken from the individual at the same time or atdifferent times. For example, samples from a tumorous portion of anorganism can be taken at different times, and used to determinedifferences in at least one species in each of the samples as tools foranalysis of the progression of a tumor.

Similarly, single species or multiple species can be used as markers.Multiple species from a single sample can be identified as separatemarkers for a particular condition, and during analysis separate speciescan be studied. As one example, a single species can define a markeridentified by or studied in connection with a single partitioningsystem. In another embodiment, multiple species from a single sample canbe identified as separate markers for a particular condition, and duringanalysis separate species can be studied. Alternatively or in addition,multiple partitioning systems can be used to study behavior of a singlespecies versus its corresponding species (markers). Or, multiple speciescan be studied and/or identified as markers in a single system ormultiple species can be identified and/or studied in connection withmultiple partitioning systems.

According to one aspect of the present invention, a computer and/or anautomated system is provided able to automatically and/or repetitivelyperform any of the methods described herein. As used herein, “automated”devices refer to devices that are able to operate without humandirection, i.e., an automated device can perform a function during aperiod of time after any human has finished taking any action to promotethe function, e.g. by entering instructions into a computer. Typically,automated equipment can perform repetitive functions after this point intime. One specific example of a technique that can make use of acomputer or other automated system is in a process in which aphysiological condition of a system as determined by determining arelative measure of interaction between one or more species from asample from the system and various interacting components of apartitioning system. In the clinical setting, this may be accomplishedby drawing a sample of blood (milliliter-sized or a very small samplesuch as a drop or less) and subjecting the blood sample or a subsetthereof (e.g., plasma) to a multi-phase partitioning process. Theresults of this process can then be compared to similar behavior ofmarkers in a similar system, which can take the form of data storedelectronically.

FIG. 3 is a schematic block diagram of an example system according toone embodiment of the present invention. In the embodiment illustratedin FIG. 3, a controller 200 is implemented on a conventional personalcomputer 250 that includes a processor 251, a memory 252, an inputdevice 253, optionally a removable storage device 254, a pointing device255, a display device 256, and a communication device 257, all coupledtogether via a bus 258. In a conventional manner, memory 252 may includea variety of memory devices, such as hard disk drives or optical diskdrives, RAM, ROM, or other memory devices and combinations thereof, andinput device 253 may include a keyboard, a microphone, or any other formof input device capable of receiving one or more inputs 210 from a userof controller 200. Removable storage device 254 may include a CD-ROMdrive, a tape drive, a diskette drive, etc. and may be used to loadapplication software, including software to implement variousembodiments of the present invention described herein. Display 256 mayinclude a conventional CRT display screen, a flat panel display screen,or any other type of display device that allows textual information tobe displayed to the user, and pointing device 255 may include a puck, ajoystick, a trackball, a mouse, or any other type of pointing device orscrolling device that permits the user to select from among the varioustextual information displayed on the display device 256. Communicationdevice 257 may include any form of communication transceiver capable ofreceiving one or more inputs 220 from the fluid-handling apparatus 30and providing one or more outputs to the fluid-handling apparatus 30.For example, communication device 257 may include a RS232/485communication transceiver, a 4-20 mA analog transceiver, an Ethernettransceiver, etc.

Software, including code that implements embodiments of the presentinvention, may be stored on some type of removable storage media such asa CD-ROM, tape, or diskette, or other computer readable mediumappropriate for the implemented memory 252 and the removable storagedevice 254. The software can be copied to a permanent form of storagemedia on the computer 250 (e.g., a hard disk) to preserve the removablestorage media for back-up purposes. It should be appreciated that inuse, the software is generally and at least partially stored in RAM, andis executed on the processor 251.

Various embodiments of the present invention can also be implementedexclusively in hardware, or in a combination of software and hardware.For example, in one embodiment, rather than a conventional personalcomputer, a Programmable Logic Controller (PLC) is used. As known tothose skilled in the art, PLCs are frequently used in a variety ofprocess control applications where the expense of a general purposecomputer is unnecessary. PLCs may be configured in a known manner toexecute one or a variety of control programs, and are capable ofreceiving inputs from a user or another device and/or providing outputsto a user or another device, in a manner similar to that of a personalcomputer. Accordingly, although embodiments of the present invention aredescribed in terms of a general purpose computer, it should beappreciated that the use of a general purpose computer is exemplaryonly, as other configurations may be used.

As shown in FIG. 3, the controller 200 is adapted to be coupled to afluid handling apparatus 30, to control operation of the fluid handlingapparatus. Controller 200 includes an input 210 to receive one or moreparameters from a user of the controller 200 relating to the desiredoperation to be performed. The controller 200 also includes a pluralityof inputs 220 to receive signals relating to the operational status ofthe fluid handling apparatus, and a plurality of outputs 230, 240 toconfigure and control the fluid handling apparatus. User inputparameters received on input 210 may include the type and amount ofprotein and/or other biomolecules that is to be processed by the fluidhandling apparatus, the compositions of liquids used by the fluidhandling apparatus for, e.g., liquid-liquid partitioning, etc.

Some embodiments of the present invention permit the user to specify oneor a number of input parameters relating to the operation of the fluidhandling apparatus, and then, based upon the input parameters, toconfigure and control the fluid handling apparatus. Depending upon thenumber of input parameters specified by the user, the controller mayprompt the user for additional parameters prior to configuring the fluidhandling apparatus.

Inputs 220 of controller 200 are adapted to receive a plurality ofsignals relating to the operational status of the fluid handlingapparatus. Signals that may be received on inputs 220 generallycorrespond to physical conditions within the fluid handling apparatus,and may include, for example, the concentration of proteins or othermolecules within the fluid handling apparatus, the time of exposure, thetime for settling to occur, the degree of agitation, the operatingtemperature or pressure, etc.

Outputs 230, 240 of the controller 200 are adapted to configure andcontrol the fluid handling apparatus, based upon the user parametersreceived at input 210, and optionally, one or more of the signalsreceived on inputs 220. Output 230 may provide a number of separatesignals, for example, a signal to introduce a protein or other moleculewithin a liquid, a signal to control the operating temperature, etc.

According to another embodiment of the present invention, controller 200may include a database and/or a knowledgebase that can be accessed byprocessor 251. According to one embodiment of the present invention, thedatabase may include a plurality of records, each record correspondingto a particular set of parameters for which the fluid processingapparatus may be used to determine a relative measure of interaction.Unless specifically indicated otherwise, as used hereinafter, the term“parameters” is used to refer to both process parameters (e.g., theamount of protein or other biomolecule(s) to be added, the operatingtemperature etc.), as well as characteristics (e.g., concentration,separation time, etc.) of the experiment given a particular set ofprocess parameters. In general, each of the records stored in thedatabase reflects empirical data based upon use of the fluid processingapparatus under defined conditions, or the use of a similar fluidprocessing apparatus under defined conditions. The controller 200 andthe database may thus be viewed as forming an “expert” system. Thedatabase may be stored on a removable storage medium and copied tomemory 252 for use by the processor 251, or alternatively, thecontroller may be pre-configured to include the database.

As will be described further below, the database (or knowledgebase) maybe configured for a particular type of fluid handling apparatus (e.g., aspecific model from a particular manufacturer of fluid handlingapparatus), or may be configured to be used with a variety of types offluid handling apparatuses. In some cases, the database may beconfigured for a particular type of protein and/or other biomolecule.Alternatively, a more general database may be used that includes anumber of different proteins, biomolecules, aqueous solutions, etc. withwhich a variety of different fluid handling apparatuses may be used. Inuse, the database may be accessed by a fluid handling apparatusconfiguration and control routine that is performed by controller 200 toconfigure and control fluid handling apparatus 30 that is operativelycoupled thereto. It should be appreciated that while the database orknowledgebase is initially based on empirical data obtained with similarequipment, the database may be periodically updated (e.g., new recordsmay be added and/or existing records may be modified) to reflectadditional data obtained in use, or by use of similar equipment.

The techniques and apparatus described herein can be used to discovermarkers or to execute a diagnostics test, in some aspects of theinvention. The apparatus could be interfaced to other devices andinstruments known to those skilled in the art, including automatedsample preparation instruments, automated immunoanalyzers, etc. Dataobtained from such devices and instruments could be electronicallychanneled to a software for performing data reduction and analysis andfor delineating a diagnostics.

The following examples are set forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of examplesof how the compounds, compositions, articles, devices and/or methodsclaimed herein are made and evaluated, are intended to be purelyexemplary of the invention, and are not intended to limit the scope ofwhat is to be regarded as the complete invention. Efforts have been madeto ensure accuracy with respect to numbers (e.g., amounts, temperature,etc.), but some errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, temperature is in ° C.or is at ambient temperature, and pressure is at or near atmospheric.The following examples are intended to illustrate certain aspects ofcertain embodiments of the present invention, but do not exemplify thefull scope of the invention.

Example 1

In this example, it was demonstrated that partitioning of a mixture canbe used to or assist in revealing structural changes in proteins.

Human serum albumin (fatty acid and gamma-globulins free), concanavalinA, cytochrome c from horse heart, beta-lactoglobulin A from bovine milk,beta-lactoglobulin B from bovine milk, ribonuclease B from bovinepancrease, lysozyme from chicken egg white, o-phthaldialdehyde reagent(complete), and Bradford Reagent were purchased from Sigma ChemicalCompany (St. Louis, Mo., USA) and used without further purification. Astock solution of a mixture of 25.6 mg albumin, 12.9 mg concanavalin A,8.2 mg cytochrome c, 12.2 mg ribonuclease B, 8.4 mg ofbeta-lactoglobulin A, and 11.5 mg beta-lactoglobulin B was prepared bydissolving in 40 ml of water. Lysozyme in the amount of 2.1 mg wasdissolved in 8 ml of this stock solution. Relative measures ofinteraction of species in these mixtures were determined by subjectingthese protein solutions to partitioning in a series of different aqueoustwo-phase systems as indicated below.

One aqueous two-phase system contained 12.16 wt % Dextran-69 (molecularweight of about 69,000), 6.05 wt % PEG-6000 (molecular weight of about6,000), 0.43 M NaClO₄, and 0.01 M sodium phosphate buffer (pH 7.4). Eachsystem was prepared by mixing the appropriate amounts of stock polymer,salt, and buffer solutions dispensed by liquid handling workstationHamilton ML-2200 into a 1.2 mL microtube. A total volume of 740microliters was dispensed to the microtube. A varied amount (0, 24, 48,72, 96, and 120 microliters) of the protein stock solution or that ofthe lysozyme solution in the protein stock solution and thecorresponding amount (120, 96, 72, 48, 24, and 0.0 microliters) of waterwere added to a system. The ratio between the volumes of the two phasesof each system of a final volume of 1.00 mL was as 1:1. The system wasshaken vigorously and then centrifuged for 30 min at about 1700 rpm tospeed resolution of the two phases. Tubes were then taken from thecentrifuge, aliquots of a given volume (20 microliters forfluorescence-based assay analysis and 10 microliters for Bradford assayanalysis) from the top and the bottom phases were withdrawn induplicate. Each aliquot was diluted, mixed with appropriate reagents,and used for the concentration analysis as described below.

The total protein concentrations in each phase were assayed by measuringthe relative fluorescence intensity in wells of a 96-well microplatewith a fluorescence microplate reader Bio-Tek F-6000 using an excitationfilter at 360 nm and an emission filter at 460 nm. For this purpose,aliquots of 20 microliters from the top and the bottom phases werewithdrawn, each mixed with 50 microliters water and 250 microliterso-phthaldialdehyde reagent, and placed into wells of a microplate forfluorescence measurements. The measured fluorescence intensities of thealiquots from the top phases were plotted as a function of thefluorescence intensities of the aliquots from the bottom phases. Thepartition coefficient for a given protein mixture was determined as aslope of the linear curve representing the plot.

In addition, the protein concentrations in each phase were assayed bymeasuring the optical absorbance at 595 nm with a using a SpectramaxPlus³⁸⁴ (Molecular Probes) UV-VIS spectrophotometer reader. For thispurpose aliquots of 10 microliters from the top and the bottom phaseswere withdrawn, each mixed with 50 microliters water and 260 microlitersBradford Reagent and placed into wells of a microplate for opticalabsorbance measurements. The mixtures were shaken for 15 min at 37° C.,and the optical absorbance at 595 nm was measured in each well. Theoptical absorbance at 595 nm of the aliquots from the top phases wereplotted as a function of the optical absorbance at 595 nm of thealiquots from the bottom phases (individual data not shown). Thepartition coefficient for the protein was determined as a slope of thelinear curve representing the plot.

The partition experiments were carried out in duplicate. The overallpartition coefficient values determined with two different assays agreedwithin 2-3% error range in each case. The individual partitioncoefficients of each protein in the mixture were determined in separateexperiments performed with individual proteins and are shown in Table 1.

TABLE 1 K-Value for K-Value for individual protein individual proteinProtein Composition Mixture A* Mixture B Albumin 2.60 2.60 ConcanavalinA 0.33 0.33 Cytochrome c 0.04 0.04 Hemoglobin 4.27 4.27beta-Lactoglobulin A 0.25 0.25 beta-Lactoglobulin B 0.32 0.32 Lysozyme6.74 6.74 Ribonuclease (A or B) 2.93 0.95

Typically, these partition coefficients would not be available a priori.Rather, partitioning of the entire mixture in the aqueous two-phasesystem would result in partitioning of individual proteins according totheir structural details. Following the mixture partitioning, standardseparation techniques, such as two-dimensional electrophoresis, could beused to quantify the concentrations of each individual protein in thetop and bottom phases of the aqueous system for each of the mixtures.The partition coefficients of each separated species can then becalculated from its concentration values in the top and bottom phasesand results similar to Table 1 would be obtained. However, separation ofthe mixtures based on size and charge alone using standard proteomicstechniques would not have shown variations since the total concentrationof each species did not change in the two mixtures. Instead, in thiscase, only the structure of a single protein was altered (in this case,glycosylation pattern).

This example thus illustrates that structural changes to individualproteins are detectable, even if their concentrations remained the samein the various mixtures. Using one or more partitioning systems thatoffer sensitivity towards different structural variations, followed bytotal mixture partitioning and standard separation techniques anddetermination and comparison of the individual partition coefficients,can be used to reveal changes to proteins that are not possible withconventional separation techniques. Furthermore, these changes couldreadily be detected and used for diagnostics screening using techniquesdescribed herein.

Example 2

In this example, it was demonstrated that the overall partitioncoefficients of total human plasma proteins from patients with aparticular disorder, as compared to healthy donors, are different underparticular partition conditions, and that this can serve as a basis fordetermination of physiological conditions of biological systems. It wasalso demonstrated that these conditions may be used for fractionation ofthe plasma for further analysis of the plasma fractions by a standardproteomics approach. The overall procedure can then be used fordiscovery of particular proteins differing in amount and/or structure inthe original samples. These proteins can also subsequently be used asmarkers specific to the disorder. This example does not intend toprovide definite data and does not define a definite procedure fordiscovering markers that underlie the particular clinical conditiondescribed; rather, it should serve as an illustrative example only.

Human plasma samples were obtained from several patients withposttraumatic stress disorder (used as experimental samples) and fromseveral people with the similar combat experience but withoutposttraumatic stress disorder (used as reference samples). One samplefrom each set (experimental and reference) was selected at random, andused for screening experiments as described below. Portions of thesamples were diluted ca. 30-fold with water, and subjected topartitioning in a variety of aqueous two-phase systems. Several systemswere found to display the different overall distribution of total plasmaproteins from the samples under comparison.

One aqueous two-phase system (PEG-Na₂SO₄) contained 15.70 wt % PEG-600(molecular weight of about 600), 9.47 wt % Na₂SO₄, and 2.30 wt %sodium/potassium phosphate buffer (pH 7.4). Each system was prepared bymixing the appropriate amounts of stock polymer, salt, and buffersolutions dispensed by liquid handling workstation Hamilton ML-2200 intoa 1.2 mL microtube. A total volume of 670 microliters was dispensed tothe microtube. A varied amount (0, 30, 60, 90, 120, and 150 microliters)of the diluted plasma sample and the corresponding amount (190, 160,130, 100, 70, and 40.0 microliters) of water were added to a system. Theratio between the volumes of the two phases of each system of a finalvolume of 0.86 mL was as 1:1. The system was shaken vigorously and thencentrifuged for 30 min at about 1700 rpm to speed resolution of the twophases. Tubes were then taken from the centrifuge, and aliquots of afixed volume from the top and the bottom phases were withdrawn induplicate.

The protein concentrations in each phase were assayed by measuring theoptical absorbance at 595 nm using a Spectramax Plus³⁸⁴ (MolecularProbes) UV-VIS spectrophotometer reader. For this purpose, aliquots of10 microliters from the top and the bottom phases were withdrawn, eachmixed with 50 microliters water and 260 microliters Bradford Reagent andplaced into wells of a microplate for optical absorbance measurements.The mixtures were shaken for 15 min at 37° C., and the opticalabsorbance at 595 nm was measured in each well. The optical absorbanceat 595 nm of the aliquots from the top phases were plotted as a functionof the optical absorbance at 595 nm of the aliquots from the bottomphases (individual data not shown). The partition coefficient for theprotein was determined as a slope of the linear curve representing theplot.

Another aqueous two-phase system (Dex-PEG) contained 11.32 wt %Dextran-69 (molecular weight of about 69,000), 14.57 wt % PEG-600(molecular weight of about 600), and 0.14 M sodium phosphate buffer (pH7.4). Each system was prepared by mixing the appropriate amounts ofstock polymer, salt, and buffer solutions dispensed by liquid handlingworkstation Hamilton ML-2200 into a 1.2 mL microtube. A total volume of620 microliters was dispensed to the microtube. A varied amount (0, 30,60, 90, 120, and 150 microliters) of the diluted plasma sample and thecorresponding amount (180, 150, 120, 90, 60, and 30.0 microliters) ofwater were added to a system. The ratio between the volumes of the twophases of each system of a final volume of 0.80 mL was as 1:1. Thesystem was shaken vigorously and then centrifuged for 30 min at about1700 rpm to speed resolution of the two phases. Tubes were then takenfrom the centrifuge, and aliquots of a fixed volume from the top and thebottom phases were withdrawn in duplicate.

The protein concentrations in each phase were assayed by measuring theoptical absorbance at 595 nm using a Spectramax Plus³⁸⁴ (MolecularProbes) UV-VIS spectrophotometer reader. For this purpose, aliquots of10 microliters from the top and the bottom phases were withdrawn, eachmixed with 50 microliters water and 260 microliters Bradford Reagent andplaced into wells of a microplate for optical absorbance measurements.The mixtures were shaken for 15 min at 37° C., and the opticalabsorbance at 595 nm was measured in each well. The optical absorbanceat 595 nm values of the aliquots from the top phases were plotted as afunction of the optical absorbance at 595 nm values of the aliquots fromthe bottom phases. The partition coefficient for the protein wasdetermined as a slope of the linear curve representing the plot.

The results of analysis of overall distribution of total plasma proteinsin the two indicated systems are presented in Table 2, which shows theoverall partition coefficients of total plasma proteins from fourpatients with posttraumatic stress disorder (PTSD, represented by thefour rows under that heading for each of the two systems) and threehealthy donors, represented by the three rows under that heading foreach of the two systems, in aqueous two-phase systems.

TABLE 2 Overall Partition Coefficient, K_(Σ) System Patients with PTSDHealthy donors PEG-Na₂SO₄ 4.16 ± 0.05 3.03 ± 0.07 4.42 ± 0.10 3.62 ±0.05 3.92 ± 0.09 2.79 ± 0.08 3.98 ± 0.13 Dex-PEG 2.93 ± 0.05 2.74 ± 0.063.54 ± 0.06 2.99 ± 0.08 3.41 ± 0.05 3.05 ± 0.05 3.02 ± 0.08

The data presented in Table 2 thus indicate that the overalldistribution of total plasma proteins differs between the samples frompatients with posttraumatic stress disorder and samples from healthydonors in the aqueous PEG-Na₂SO₄ two-phase system to a larger extentthan in the aqueous Dex-PEG two-phase system. Therefore, the formersystem was used for fractionation of the samples by extraction.

Aqueous PEG-Na₂SO₄ mixtures of total final system volume 4.0 ml eachwere prepared. Plasma samples (undiluted) of 0.3 ml volume each wereadded to the mixtures, vortexed, and centrifuged as described above.Following settling of the phases, aliquots of about 1.5 ml volume werewithdrawn from the upper phases and 2-D HPLC analysis of the extractswas performed. The 2-D HPLC analysis included two separation steps. Thefirst step was chromatofocusing separation was performed using the HPCF1D column (Eprogen, Darien, Ill.) with a flow rate of 0.2 ml/min, abuffer gradient from pH 8.5 to pH 4.0 and detection at 280 nm. The pH ofthe eluate was monitored and the fractions of proteins within certain pHranges are collected. The fractions collected from the first dimensionHPLC separation were then analyzed in the second dimension byreversed-phase HPLC (RP-HPLC). Reversed-phase HPLC separation wasperformed using a nonporous HPRP-2D column (Eprogen, Darien, Ill.) at50° C. with flow rate of 0.75 ml/min, acetonitrile gradient from 0 to100% in 30 min and detection at 214 nm. The injection volume of eachfraction obtained in the first procedure was 500 microliters.

The typical results of the 2-D HPLC analysis are presented in FIGS. 1and 2. FIG. 1 shows the RP-HPLC chromatogram of proteins selected fromthe above samples that had a pI range of 3.5<pI<3.9. The chromatogram ofthe extract from control plasma sample is indicated as 10; thechromatogram of the extract from the PTSD sample is indicated as 20. Thepeaks in the PTSD sample with retention times that were different fromthose observed in the control plasma sample are denoted with arrows withquestion marks in FIG. 1. Peaks which appeared in the PTSD sample butwere absent from the control sample are denoted by arrows. FIG. 2 showsa similar RP-HPLC chromatogram analysis of selected proteins having a pIfrom 4.3 to 4.6. All the marks as indicated with respect to FIG. 1.

These results illustrate that the protein patterns of extracts from thesamples under comparison were found to be different. These differencesaccording to the chromatograms shown in FIGS. 1-2 include differentrelative amounts of the proteins (displayed as different relativeheights of the peaks on chromatograms), different structures of theproteins (displayed as different retention times or positions of thepeaks on chromatograms), and the appearance and/or lack of certainproteins in the samples under comparison (displayed asappearance/disappearance of peaks on chromatograms).

As a hypothetical (prophetic) section of this example, a more definitivestudy for discovering markers under the procedures described herein caninclude performing the same 2-D HPLC assays on the other phases of thepartitioning systems, to be followed by calculation of the individualpartition coefficients of each peak. Differences between coefficientscorresponding to each peak in the two cases of healthy vs. positive thendefine each peak as a potential marker. The marker can then be used forsubsequent diagnostics of unknown samples in the manner describedherein.

This demonstrates the ability of the invention to be effective evenwithout determining the chemical or biological identity of specificspecies that are analyzed in (e.g., via study of relative measures ofinteraction) and used in connection with the invention.

Thus, this example illustrates that the analysis of the overall proteindistribution in a particular aqueous two-phase system displayeddifferent distribution behavior of total plasma proteins from blood ofpatients with a certain disease and from blood of healthy donors. It wasfound that there were different overall distributions of total plasmaproteins from the samples under comparison. As described above, furtheranalysis of the fractions by proteomics analysis can also be performed,and further analysis and calculation of the individual partitioncoefficients of certain peaks of interest that are different between thesamples could be performed and used for diagnostics screening asdescribed by the present invention. Alternatively, site-specificantibodies could be developed against proteins that underlie certainpeaks showing differences, thus bypassing the need for partitioning inthe screening stage of such markers.

Example 3

In this example, it was demonstrated that relative measures ofinteraction, exemplified herein as the partition coefficients of aqueoustwo-phase partitioning systems, of certain human serum proteins frompatients with a particular physiological condition, were different thanthose corresponding to healthy donors, and that such differences couldserve as a basis for determination of physiological conditions ofbiological systems. Selection of partitioning conditions suitable fordiscovering changes in the structure of certain proteins, and/or intheir interactions with other proteins in serum, was also illustrated.Furthermore, this example illustrates one method of identifyingparticular proteins as potential biomarkers from a group of proteins inbiological fluids using methods described in the present invention.

Serum samples from patients diagnosed with early stage ovarian cancerand healthy women were obtained from Gynecologic Oncology Group (GOG,Columbus, Ohio). The 250 microliters aliquots of sera from each of 10patients with ovarian cancer were combined and mixed, and used as apool. The 250 ul microliter aliquots of sera from 10 healthy women werealso combined and mixed, and used as a pool. Portions of 75 microlitersvolume from each pooled sample were subjected to partitioning in avariety of 24 aqueous two-phase systems.

Each aqueous partitioning system was prepared by mixing the appropriateamounts of stock polymer, salt, and buffer solutions dispensed by liquidhandling workstation Hamilton ML-4000 into a 1.2 mL microtube. Forexample, one aqueous two-phase system (Dex-Ficoll) contained 18.00 wt %Ficoll-70 (molecular weight of about 70,000), 13.00 wt % Dextran-75(molecular weight of about 75,000), 1.00 wt % sodium chloride, and 0.01M sodium/potassium phosphate buffer (pH 7.4). A total volume of 500microliters was dispensed to the microtube. A fixed amount of 75microliters of pooled serum sample and 75 microliters of water wereadded to a system. The ratio between the volumes of the two phases ofeach system of a final volume of 0.65 mL was as 1:1. The system wasshaken vigorously and then centrifuged for 60 min at about 1700 rpm tospeed resolution of the two phases. Tubes were then removed from thecentrifuge, and aliquots of a fixed 100 microliters volume from the topand the bottom phases were withdrawn. Each aliquot was diluted 5-foldwith water and stored at −80° C. The same procedure was followed withboth serum samples from pooled sample of sera from patients with ovariancancer and from pooled sample of sera from healthy women. The sameprotocol was also used for partitioning in all of the different aqueoustwo-phase systems screened.

The frozen diluted aliquots from top and bottom phase of each aqueoustwo-phase system used for screening were coded and shipped to LuminexCore Facility at Hillman Cancer Center (Pittsburgh, Pa.), where standardLabMap assays (Bio-Rad Laboratories, Hercules, Calif.) were utilized formeasuring concentrations of certain proteins, such as interleukin(IL)-8, vascular endothelial growth factor VEGF, basic fibroblast growthfactor (bFGF), tumor necrosis factor alpha (TNF-alpha), tumor necrosisfactor receptor (TNF-R1), granulocyte colony-stimulating factor (G-CSF),and others. The LabMap assays were performed in 96-well microplateformat according to appropriate manufacturer's protocols. Samples wereanalyzed using the Bio-Plex suspension array system (Bio-RadLaboratories, Hercules, Calif.) as described in the literature. See, forexample, Gorelik et al., Cancer Epidimiol. Biomarkers Prev., 14(4),981-987 (2005). Analysis of experimental data was performed usingfive-parametric-curve fitting.

The partition coefficient for each individual protein in each aqueoustwo-phase system was calculated as the ratio of the proteinconcentration assayed in the top phase to that in the bottom phase. Theconcentrations of 65 proteins out of about 80 different proteins whichassayed in both phases of 24 different aqueous two-phase systems couldnot be reliably assayed in one or both phases of at least some ofaqueous two-phase partitioning systems. In other cases, the partitioncoefficient could be determined, for example, for pooled serum fromovarian cancer patients but not for serum from healthy women. Thesecases were eliminated from further consideration.

In many cases, individual serum proteins the partition coefficients weredetermined to be statistically indistinguishable, within theconcentration assay error margin, between sera samples from cancerpatients and healthy women. Such cases denote either of the followinginterpretations: (1) there were no structural changes for the particularprotein under consideration between normal and cancer samples; (2) theparticular aqueous partitioning system was not suitable for identifyingstructural changes in the particular protein under examination. In theformer case, the protein could be rejected as a potential marker, whilein the latter case the partitioning system could be rejected as beingunsuitable for discerning differences in the specific protein. Thus thecombination of protein and system could be rejected for denoting adifference in a physiological condition between the samples.

For a number of individual proteins, however, it was possible toestablish the partition conditions under which the changes in theprotein structure and/or protein interactions with other proteins wereobserved between the cancer and normal sera samples as significantdifferences between the partition coefficient values, K, for a givenprotein. The results obtained for several protein markers and partitionconditions used are presented in Table 3.

TABLE 3 Partitioning K (serum from K (serum from cancer System*Protein** healthy women) patients) A EGFR 5.11 6.94 H 0.39 0.68 A IL-85.35 4.14 G 7.05 4.18 D 5.60 4.34 E G-CSF 0.68 0.15 F 1.06 1.63 B 0.750.24 C 0.29 0.02 B sIL-6R 1.92 1.05 A IGFBP-1 5.13 3.93 A bFGF 0.41 1.04A RANTES 3.34 1.97 *Partitioning Systems: A: 18.0 wt % Fcioll-70, 13.0wt % Dextran-75, 2.2 wt % K/Na phosphate buffer, pH 7.4; B: 8.0 wt %Fcioll-70, 13.0 wt % Dextran-75, 1 wt % NaCl, 0.15 wt % K/Na phosphatebuffer, pH 7.4; C: 18.0 wt % Fcioll-70, 13.0 wt % Dextran-75, 3.8 wt %NaCl, 0.15 wt % K/Na phosphate buffer, pH 7.4; D: 15.7 wt % PEG-600, 1.0wt % NaCl, 18.1 wt % K/Na phosphate buffer, pH 7.4; E: 15.7 wt %PEG-600, 9.5 wt % Na₂SO₄, 1.0 wt % NaCl, 0.64 wt % K/Na phosphatebuffer, pH 7.4; F: 15.7 wt % PEG-600, 9.5 wt % Na₂SO₄, 4.8 wt % NaCl,0.64 wt % K/Na phosphate buffer, pH 7.4; G: 15.7 wt % PEG-600, 9.5 wt %Na₂SO₄, 3.8 wt % NaCl, 2.3 wt % K/Na phosphate buffer, pH 7.4; H: 15.7wt % PEG-600, 9.5 wt % Na₂SO₄, 1.0 wt % NaCl, 2.3 wt % K/Na phosphatebuffer, pH 7.4; **Proteins: EGFR: epidermal growth factor receptor;IL-8: interleukin-8; G-CSF: granulocyte colony-stimulating factor;sIL-6R: soluble interleukin-6 receptor; IGFBP-1: insulin-like growthfactor binding protein 1; bFGF: basic fibroblast growth factor; RANTES:beta-chemokine (regulated upon activation, normal T-cell expressed andsecreted).

This example demonstrates certain aspects, including the following: (1)partitioning systems may be selected or rejected as tools to determinechanges in physiological conditions; (2) individual proteins may beselected or rejected as potential markers for same application; (3)specific combinations of protein and systems may be used individually orin tandem to screen individuals for changes in physiological conditions.Screening of individuals using biomarkers discovered according to somemethods described in the present invention may be accomplished after atleast one combination of a partitioning system and a protein aredesignated as suitable for establishing differences in physiologicalconditions of clinical relevance. Such screening could be accomplished,using the present example, in several principal manners (additional waysmay be defined by those skilled in the art). One manner relies on usinga single protein and a single system, e.g., EGFR with system A, or EGFRwith system H, or IL-8 with system G. Actual clinical use of a singleprotein and system as a biomarker suitable for diagnosing a particulardisease may involve further procedures to establish its sensitivity andspecificity using techniques known to those skilled in the art. Anothermanner relies on using a single partitioning system and multipleproteins. For example, a clinical diagnostic assay may comprise serumpartitioning in a single aqueous two-phase system followed byimmunoassay analysis of several individual proteins of clinical value,for example, system A in Table 3, for analysis of changes in partitioncoefficients for, e.g., EGFR, IL-8, IGFBP-1, bFGF, and RANTES. Thismanner might be especially useful since it requires a singlepartitioning step of serum, followed by a multiplexed immunoassay. Theuse of multiple protein markers for diagnosis can increase thesensitivity and specificity trade-off and is recognized by those skilledin the art. Yet another manner in which screening may be efficientlyaccomplished is using different systems and proteins to arrive at adesired statistical performance parameters. For example, differentaqueous two-phase systems could be used for analysis of changes inpartition coefficients of different individual proteins, e.g., system Hfor EGFR, system G for IL-8, system E for GT-CSF, system B for sIL-6R,system A for bFGF, RANTES, and IGFBP-1, etc. Still another manner mayinvolve using several aqueous two-phase systems for analysis ofpartition coefficients of a single protein.

Example 4

This example illustrates that partitioning of prostate specific antigen(PSA) in urine may be independent of the total PSA concentration inurine and serum.

Urine samples were collected from patients who were candidates forprostate biopsy based on serum PSA levels above 4 ng/ml, or based onother clinical presentations, such as pelvic pain and voidingdisfunction or other physiological abnormalities. The urine samples werecollected immediately post prostate massage, a clinical protocolperformed by a physician, and included 4 passes over the prostate withpressure lasting at least 5 seconds. Each urine sample was transferredinto 5 ml polypropylene tube, centrifuged for 10 min at 3,000 rpm,supernatant separated, aliquoted by 0.5 ml, placed into Eppendorfmicrotubes, and stored at −80° C. until further use. Urine samples from9 patients with prostate cancer as established by prostate biopsyanalysis were thawed and combined to form a “cancer” urine pool sample.Urine samples from patients established to be free of prostate canceraccording to results from prostate biopsy analysis were thawed andcombined to form a “control” urine pool sample. These pool samples werestored at −80° C. until further use.

Dextran-75 (with molecular weight of 60,000 to 90,000) was purchasedfrom USB Corporation (Cleveland, Ohio, USA) and used without furtherpurification. Ficoll-70 (with molecular weight of about 70,000) waspurchased from GE Healthcare Bio-Sciences Corp. (Piscataway, N.J., USA)and used without further purification. All inorganic salts of ACSreagent grade were purchased from Sigma Chemical Company (St. Louis,Mo., USA) and used without further purification. Stock solutions ofindividual polymers and inorganic salts in water were preparedgravimetrically and used to form aqueous two-phase system as describedbelow.

The aqueous two-phase system contained 18.00 wt % Ficoll-70, 13.00 wt %dextran-75, and 0.15 M NaCl in 0.010 M sodium/potassium phosphate buffer(pH 7.4). Each system was prepared by mixing the appropriate amounts ofstock polymer, salt and buffer solutions by weight into a 1.2 mLmicrotube up to a total weight of a system of 0.426 g (volume 0.369 mL)using a MICROLAB 4000 MPH-4 liquid handling robotics workstation(Hamilton Company, Reno, Nev., USA). Urine samples from individualpatients in amounts of 0.075 mL were added to each system. The systemswere vigorously shaken by vortexing and centrifuged for 60 min at about2500 rpm in a refrigerated centrifuge set to room temperature andequipped with a microplate rotor to speed the phase settling. Themicrotubes were taken out of the centrifuge, and aliquots of 100microliter from the top and the bottom phases were withdrawn and eachdiluted with 0.400 ml water for PSA concentration analysis.

PSA concentration was determined in each aliquot by an automatedenzyme-linked immunosorbent assay (ELISA), Elecsys Total PSA Immunoassaywith a lower detection limit of 0.002 ng/ml (Roche Diagnostics,Indianapolis, Ind., USA).

The results obtained are plotted in FIG. 4 as the PSA concentrations fordifferent individual patients determined in the top Ficoll-rich phaseversus the PSA concentrations for the same individual patientsdetermined in the bottom dextran-rich phase. The data given in FIG. 4indicated a linear continuous relationship between the PSAconcentrations measured for different patients in the upper phase andPSA concentrations for the same patients measured in the bottom phase.The slope of this linear relationship represents the ratio of the PSAconcentration in the top phase to that in the bottom phase for eachindividual patient, which is the PSA relative measure of interactionwith the two phases, here referred to as the PSA partition coefficient.These data indicated that the PSA partition coefficient for any specificpatient is independent of the total PSA expression level (orconcentration) for the same individual. Therefore, if the overallconcentration level of PSA is dependent upon physiological conditionsother than cancer, for example, a benign prostate condition, itsspecificity as prostate cancer marker may be reduced (as is the case forPSA). The independence of the PSA relative measure of interaction fromits concentration level could be useful for increasing the cancerspecificity of PSA, if it has structural or other properties specific tocancer that could be expressed via changes to its relative measure ofinteraction with systems according to techniques described herein.

Example 5

This example illustrates partitioning of PSA from urine from patientswith prostate cancer in aqueous PEG-salt two-phase system differs fromthat of PSA from urine from patients free of prostate cancer.

Urine samples were collected from patients who were candidates forprostate biopsy based on serum PSA levels above 4 ng/ml or based onother clinical presentations. The urine samples were collectedimmediately post prostate massage performed by a physician and included4 passes over the prostate with pressure lasting at least 5 seconds.Each urine sample was transferred into 5 ml polypropylene tube,centrifuged for 10 min at 3,000 rpm, supernatant separated, aliquoted by0.5 ml, placed into Eppendorf microtubes, and stored at −80° C. untilfurther use. Urine samples from 9 patients with prostate cancerestablished by prostate biopsy analysis were thawed and combined to forma “cancer” urine pool sample. Urine samples from patients established tobe free of prostate cancer according to results from prostate biopsyanalysis were thawed and combined to form a “control” urine pool sample.These pool samples were stored at −80° C. until further use.

Poly(ethylene glycol)-8000 (PEG-8000) (with molecular weight of 8,000)and inorganic salts of ACS reagent grade were purchased from SigmaChemical Company (St. Louis, Mo., USA) and used without furtherpurification. Stock solutions of individual polymer and inorganic saltsin water were prepared gravimetrically and used to form aqueoustwo-phase system as described below.

The aqueous two-phase system contained 12.60 wt % PEG-8000, 8.90 wt %sodium/potassium phosphate buffer (pH 7.4), and 10.5 wt % NaCl. Eachsystem was prepared by mixing the appropriate amounts of stock polymer,salt and buffer solutions by weight into a 1.2 mL microtube up to atotal weight of a system of 0.460 g (volume 0.387 mL) using a MICROLAB4000 MPH-4 liquid handling robotics workstation (Hamilton Company, Reno,Nev., USA). Urine samples from cancer urine pool and control urine poolin amounts of 0.075 mL were added to each system. The systems werevigorously shaken by vortexing and centrifuged for 60 min at about 2500rpm in a refrigerated centrifuge at room temperature with a microplaterotor to speed the phase settling. The microtubes were taken out of thecentrifuge, and aliquots of 100 ml from the top and the bottom phaseswere withdrawn and each diluted with 0.400 ml water for PSAconcentration analysis.

PSA concentration was determined in each aliquot by an automatedenzyme-linked immunosorbent assay (ELISA), Elecsys Total PSAImmunoassay, lower detection limit 0.002 ng/ml; Roche Diagnostics(Indianapolis, Ind., USA).

The results of PSA concentration measurements in the two phases and thePSA partition coefficients calculated as the ratios of the PSAconcentration in the top phase to the PSA concentration in the bottomphase are presented in Table 4.

TABLE 4 Control urine* pool Prostate cancer urine* pool Sample PSA level12,430** ng/ml  4,560** ng/ml Top phase PSA 610.00** ng/ml 199.00**ng/ml concentration Bottom phase PSA   46.16 ng/ml   24.19 ng/mlconcentration PSA partition 13.2 8.2 coefficient, K *Urine was collectedpost prostate massage as described above; **PSA concentrations weremeasured followed additional 100-fold dilution with universal diluent(Roche Diagnostics).

The results obtained with the pooled urine samples indicated thatpartition coefficient for PSA from patients with prostate cancer in theaqueous PEG-8000-salt two-phase system used in this example differs fromthe partition coefficient of PSA from patients free of prostate cancer.Therefore, the particular system used in this example can be useful fora method for detecting prostate cancer according to techniques andmethods described in the present invention.

Example 6

This example illustrates that partitioning of PSA from urine frompatients with prostate cancer in aqueous PEG-dextran two-phase systemdiffers from that of PSA from urine from patients free of prostatecancer.

Urine samples were collected from patients who were candidates forprostate biopsy based on serum PSA levels above 4 ng/ml or based onother clinical presentations. The urine samples were collectedimmediately post prostate massage performed by a physician and included4 passes over the prostate with pressure lasting at least 5 seconds.Each urine sample was transferred into 5 ml polypropylene tube,centrifuged for 10 min at 3,000 rpm, supernatant separated, aliquoted by0.5 ml, placed into Eppendorf microtubes, and stored at −80° C. untilfurther use. Urine samples from 9 patients with prostate cancerestablished by prostate biopsy analysis were thawed and combined to forma “cancer” urine pool sample. Urine samples from patients established tobe free of prostate cancer according to results from prostate biopsyanalysis were thawed and combined to form a “control” urine pool sample.These pool samples were stored at −80° C. until further use.

Dextran-75 (with molecular weight of 60,000 to 90,000) was purchasedfrom USB Corporation (Cleveland, Ohio, USA) and used without furtherpurification. Poly(ethylene glycol)-600 (PEG-600) (with molecular weightof 600) and inorganic salts of ACS reagent grade were purchased fromSigma Chemical Company (St. Louis, Mo., USA) and used without furtherpurification. S tock solutions of individual polymers and inorganicsalts in water were prepared gravimetrically and used to form aqueoustwo-phase system as described below.

The aqueous two-phase system contained 11.90 wt % dextran-75, 15.7 wt %PEG-600, 0.15 M sodium sulfate in 0.01 M sodium phosphate buffer (pH7.4). Each system was prepared by mixing the appropriate amounts ofstock polymer, salt and buffer solutions by weight into a 1.2 mLmicrotube up to a total weight of a system of 0.433 g (volume 0.394 mL)using a MICROLAB 4000 MPH-4 liquid handling robotics workstation(Hamilton Company, Reno, Nev., USA). Urine samples from cancer urinepool and control urine pool in amounts of 0.075 mL were added to eachsystem. The systems were vigorously shaken by vortexing and centrifugedfor 60 min at about 2500 rpm in a refrigerated centrifuge at roomtemperature with a microplate rotor to speed the phase settling. Themicrotubes were taken out of the centrifuge, and aliquots of 100microliter from the top and the bottom phases were withdrawn and eachdiluted with 0.400 ml water for PSA concentration analysis.

PSA concentration was determined in each aliquot by an automatedenzyme-linked immunosorbent assay (ELISA) Elecsys Total PSA Immunoassay;lower detection limit 0.002 ng/ml; (Roche Diagnostics, Indianapolis,Ind., USA).

The results of PSA concentration measurements in the two phases and thePSA partition coefficients calculated as the ratios of the PSAconcentration in the top phase to the PSA concentration in the bottomphase are presented in Table 5.

TABLE 5 Control urine* pool Prostate cancer urine* pool Sample PSA level12,430** ng/ml 4,560** ng/ml  Top phase PSA 366.00** ng/ml 24.78 ng/mlconcentration Bottom phase PSA   20.96 ng/ml  3.62 ng/ml concentrationPSA partition 17.5 6.8 coefficient, K *Urine was collected post prostatemassage as described above; **PSA concentrations were measured followedadditional 100-fold dilution with universal diluent (Roche Diagnostics).

The results obtained with the pooled urine samples indicated thatpartition coefficient for PSA from patients with prostate cancer in theaqueous PEG-dextran two-phase system used in this example differs fromthe partition coefficient of PSA from patients free of prostate cancer.Therefore, the particular system described in the present example can beused for diagnostics purposes according to techniques described in thepresent invention.

Example 7

This example illustrates partitioning of PSA from urine from patientswith prostate cancer in aqueous dextran-Ficoll-phosphate buffertwo-phase systems of different salt compositions differs from that ofPSA from urine from patients free of prostate cancer.

Urine samples were collected from patients who were candidates forprostate biopsy based on serum PSA levels above 4 ng/ml or based onother clinical presentations. The urine samples were collectedimmediately post prostate massage performed by a physician and included4 passes over the prostate with pressure lasting at least 5 seconds.Each urine sample was transferred into 5 ml polypropylene tube,centrifuged for 10 min at 3,000 rpm, supernatant separated, aliquoted by0.5 ml, placed into Eppendorf microtubes, and stored at −80° C. untilfurther use. Urine samples from 9 patients with prostate cancerestablished by prostate biopsy analysis were thawed and combined to forma “cancer” urine pool sample. Urine samples from patients established tobe free of prostate cancer according to results from prostate biopsyanalysis were thawed and combined to form a “control” urine pool sample.These pool samples were stored at −80° C. until further use.

Dextran-75 (with molecular weight of 60,000 to 90,000) was purchasedfrom USB Corporation (Cleveland, Ohio, USA) and used without furtherpurification. Ficoll-70 (with molecular weight of about 70,000) waspurchased from GE Healthcare Bio-Sciences Corp. (Piscataway, N.J., USA)and used without further purification. All inorganic salts of ACSreagent grade were purchased from Sigma Chemical Company (St. Louis,Mo., USA) and used without further purification. Stock solutions ofindividual polymers and inorganic salts in water were preparedgravimetrically and used to form aqueous two-phase system as describedbelow.

The aqueous two-phase system contained 18.00 wt % Ficoll-70, 13.0 wt %dextran-75, 2.2 wt % (0.15 M) sodium/potassium phosphate buffer (pH7.4). Each system was prepared by mixing the appropriate amounts ofstock polymer, salt and buffer solutions by weight into a 1.2 mLmicrotube up to a total weight of a system of 0.421 g (volume 0.359 mL)using a MICROLAB 4000 MPH-4 liquid handling robotics workstation(Hamilton Company, Reno, Nev., USA). Urine samples from cancer urinepool and control urine pool in amounts of 0.075 mL were added to eachsystem. The systems were vigorously shaken by vortexing and centrifugedfor 60 min at about 2,500 rpm in a refrigerated centrifuge at roomtemperature with a microplate rotor to speed the phase settling. Themicrotubes were taken out of the centrifuge, and aliquots of 100microliter from the top and the bottom phases were withdrawn and eachdiluted with 0.400 ml water for PSA concentration analysis.

PSA concentration was determined in each aliquot by an automatedenzyme-linked immunosorbent assay (ELISA), Elecsys Total PSAImmunoassay, lower detection limit 0.002 ng/ml (Roche Diagnostics,Indianapolis, Ind., USA).

The results of PSA concentration measurements in the two phases and thePSA partition coefficients calculated as the ratios of the PSAconcentration in the top phase to the PSA concentration in the bottomphase are presented in Table 6.

TABLE 6 Control urine* pool Prostate cancer urine* pool Sample PSA level12,430** ng/ml 4,560** ng/ml  Top phase PSA 243.00** ng/ml 66.61 ng/mlconcentration Bottom phase PSA   11.48 ng/ml  4.35 ng/ml concentrationPSA partition 21.2 15.3 coefficient, K *Urine was collected postprostate massage as described above; **PSA concentrations were measuredfollowed additional 100-fold dilution with universal diluent (RocheDiagnostics).

The results obtained with the pooled urine samples indicated thatpartition coefficient for PSA from patients with prostate cancer in theaqueous dextran-Ficoll-phosphate buffer two-phase system used in thisexample differs from the partition coefficient of PSA from patients freeof prostate cancer. Therefore, the particular system described in thepresent example can be used for diagnostics purposes according totechniques described in the present invention.

Example 8

This example illustrates partitioning of PSA from urine from patientswith prostate cancer in aqueous dextran-Ficoll-sodium sulfate-phosphatebuffer two-phase systems of different salt compositions differs fromthat of PSA from urine from patients free of prostate cancer.

Urine samples were collected from patients who were candidates forprostate biopsy based on serum PSA levels above 4 ng/ml or based onother clinical presentations. The urine samples were collectedimmediately post prostate massage performed by a physician and included4 passes over the prostate with pressure lasting at least 5 seconds.Each urine sample was transferred into 5 ml polypropylene tube,centrifuged for 10 min at 3,000 rpm, supernatant separated, aliquoted by0.5 ml, placed into Eppendorf microtubes, and stored at −80° C. untilfurther use. Urine samples from 9 patients with prostate cancerestablished by prostate biopsy analysis were thawed and combined to forma “cancer” urine pool sample. Urine samples from patients established tobe free of prostate cancer according to results from prostate biopsyanalysis were thawed and combined to form a “control” urine pool sample.These pool samples were stored at −80° C. until further use.

Dextran-75 (with molecular weight of 60,000 to 90,000) was purchasedfrom USB Corporation (Cleveland, Ohio, USA) and used without furtherpurification. Ficoll-70 (with molecular weight of about 70,000) waspurchased from GE Healthcare Bio-Sciences Corp. (Piscataway, N.J., USA)and used without further purification. All inorganic salts of ACSreagent grade were purchased from Sigma Chemical Company (St. Louis,Mo., USA) and used without further purification. Stock solutions ofindividual polymers and inorganic salts in water were preparedgravimetrically and used to form aqueous two-phase system as describedbelow.

The aqueous two-phase system contained 18.00 wt % Ficoll-70, 13.0 wt %dextran-75, 0.15 M sodium sulfate, and 0.01 M sodium phosphate buffer(pH 7.4). Each system was prepared by mixing the appropriate amounts ofstock polymer, salt and buffer solutions by weight into a 1.2 mLmicrotube up to a total weight of a system of 0.431 g (volume 0.367 mL)using a MICROLAB 4000 MPH-4 liquid handling robotics workstation(Hamilton Company, Reno, Nev., USA). Urine samples from cancer urinepool and control urine pool in amounts of 0.075 mL were added to eachsystem. The systems were vigorously shaken by vortexing and centrifugedfor 60 min at about 2,500 rpm in a refrigerated centrifuge at roomtemperature with a microplate rotor to speed the phase settling. Themicrotubes were taken out of the centrifuge, and aliquots of 100microliter from the top and the bottom phases were withdrawn and eachdiluted with 0.400 ml water for PSA concentration analysis.

PSA concentration was determined in each aliquot by an automatedenzyme-linked immunosorbent assay (ELISA), Elecsys Total PSAImmunoassay, lower detection limit 0.002 ng/ml (Roche Diagnostics,Indianapolis, Ind., USA).

The results of PSA concentration measurements in the two phases and thePSA partition coefficients calculated as the ratios of the PSAconcentration in the top phase to the PSA concentration in the bottomphase are presented in Table 7.

TABLE 7 Control urine* pool Prostate cancer urine* pool Sample PSA level12,430** ng/ml  4,560** ng/ml Top phase PSA 298.00** ng/ml 159.00**ng/ml concentration Bottom phase PSA   64.95 ng/ml   13.01 ng/mlconcentration PSA partition 4.6 12.2 coefficient, K *Urine was collectedpost prostate massage as described above; **PSA concentrations weremeasured followed additional 100-fold dilution with universal diluent(Roche Diagnostics).

The results obtained with the pooled urine samples indicated thatpartition coefficient for PSA from patients with prostate cancer in theaqueous dextran-Ficoll-sodium sulfate-phosphate buffer two-phase systemused in this example differs from the partition coefficient of PSA frompatients free of prostate cancer. Therefore, the particular systemdescribed in the present example can be used for diagnostics purposesaccording to techniques described in the present invention.

Example 9

This example illustrates partitioning of PSA from urine from individualpatients with prostate cancer in aqueous dextran-Ficoll-sodiumisothiocyanate-phosphate buffer two-phase systems of different saltcompositions differs from that of PSA from urine from patients free ofprostate cancer.

Urine samples were collected from patients who were candidates forprostate biopsy based on serum PSA levels above 4 ng/ml or based onother clinical presentations. The urine samples were collectedimmediately post prostate massage performed by a physician and included4 passes over the prostate with pressure lasting at least 5 seconds.Each urine sample was transferred into 5 ml polypropylene tube,centrifuged for 10 min at 3,000 rpm, supernatant separated, aliquoted by0.5 ml, placed into Eppendorf microtubes, and stored at −80° C. untilfurther use.

Dextran-75 (with molecular weight of 60,000 to 90,000) was purchasedfrom USB Corporation (Cleveland, Ohio, USA) and used without furtherpurification. Ficoll-70 (with molecular weight of about 70,000) waspurchased from GE Healthcare Bio-Sciences Corp. (Piscataway, N.J., USA)and used without further purification. All inorganic salts of ACSreagent grade were purchased from Sigma Chemical Company (St. Louis,Mo., USA) and used without further purification. Stock solutions ofindividual polymers and inorganic salts in water were preparedgravimetrically and used to form aqueous two-phase system as describedbelow.

The aqueous two-phase system contained 18.00 wt % Ficoll-70, 13.00 wt %dextran-75, and 0.15 M NaSCN in 0.010 M sodium phosphate buffer (pH7.4). Each system was prepared by mixing the appropriate amounts ofstock polymer, salt and buffer solutions by weight into a 1.2 mLmicrotube up to a total weight of a system of 0.426 g (volume 0.369 mL)using a MICROLAB 4000 MPH-4 liquid handling robotics workstation(Hamilton Company, Reno, Nev., USA). Urine samples from individualpatients in amounts of 0.075 mL were added to each system. The systemswere vigorously shaken by vortexing and centrifuged for 60 min at about2,500 rpm in a refrigerated centrifuge at room temperature with amicroplate rotor to speed the phase settling. The microtubes were takenout of the centrifuge, and aliquots of 100 microliter from the top andthe bottom phases were withdrawn and each diluted with 0.400 ml waterfor PSA concentration analysis.

PSA concentration was determined in each aliquot by an automatedenzyme-linked immunosorbent assay (ELISA), Elecsys Total PSAImmunoassay, lower detection limit 0.002 ng/ml (Roche Diagnostics,Indianapolis, Ind., USA).

The results obtained are presented in Table 8 as the PSA concentrationsfor different individual patients measured in serum, and concentrationsdetermined in the top Ficoll-rich phase versus the PSA concentrationsfor the same individual patients determined in the bottom dextran-richphase.

TABLE 8 No prostate cancer Prostate cancer Serum total PSA Partition PSAPartition Patient # PSA, ng/ml coefficient, K value coefficient, K value 1 4.12 1.06  2 13.59 1.23  3 11.15 1.42  4 4.9 1.69   5** 7.74 1.65  6** 4.58 1.34  7 5.7 1.73 11 10.3 1.62  12** 15.6 1.61 13 6.06 1.86 146.6 1.43 15 4.25 1.68 16 6.6 1.36 17 5.1 1.44 18 3.64 1.51 19 4.65 1.6120 9.36 1.60 22 7.98 1.46 23 4.05 1.13 26 4.7 1.30 *Urine was collectedpost prostate massage as described above; **patients were established tohave benign prostate disease by prostate biopsy analysis.

The data in Table 8 were subsequently processed using standardstatistical techniques for detecting statistically significance betweencancer and normal samples, and for obtaining the relationship betweenspecificity and sensitivity. FIG. 5 depicts the ROC (Receiver-OperatorCharacteristics) curve calculated using SigmaPlot 10 statisticalsoftware (Systat Software, San Jose, Calif., USA). The data indicate adeparture from the diagonal, with area under the curve of 0.77(P=0.044). The ROC curve provides a relationship between sensitivity andspecificity for a given cut-off value in the partition coefficient. Forexample, a diagnostics test could be devised to further stratifypatients with elevated PSA levels and/or other clinical presentationsinto those who should receive biopsy and those who should be activelymonitored. For such a clinical application, a cut-off value for thepartition coefficient could be selected at 1.3, resulting in 100%specificity (or false positive fraction). If the sensitivity of the testis provided a priori by other means (e.g., PSA level, velocity and otherclinical presentations), then the test using partition coefficient asdescribed in the present invention can be used to reduce the number offalse positive biopsies.

While several embodiments of the invention have been described andillustrated herein, those of ordinary skill in the art will readilyenvision a variety of other means and structures for performing thefunctions and/or obtaining the results or advantages described herein,and each of such variations or modifications is deemed to be within thescope of the present invention. More generally, those skilled in the artwould readily appreciate that all parameters, dimensions, materials, andconfigurations described herein are meant to be exemplary and thatactual parameters, dimensions, materials, and configurations will dependupon specific applications for which the teachings of the presentinvention are used. Those skilled in the art will recognize, or be ableto ascertain using no more than routine experimentation, manyequivalents to the specific embodiments of the invention describedherein. It is, therefore, to be understood that the foregoingembodiments are presented by way of example only and that, within thescope of the appended claims and equivalents thereto, the invention maybe practiced otherwise than as specifically described. The presentinvention is directed to each individual feature, system, materialand/or method described herein. In addition, any combination of two ormore such features, systems, materials and/or methods, if such features,systems, materials and/or methods are not mutually inconsistent, isincluded within the scope of the present invention.

The definitions, as used herein, should be understood to control overdictionary definitions, definitions in documents incorporated byreference, and/or ordinary meanings of the defined terms.

It should also be understood that, unless clearly indicated to thecontrary, in any methods claimed herein that include more than one act,the order of the acts of the method is not necessarily limited to theorder in which the acts of the method are recited.

In the claims (as well as in the specification above), all transitionalphrases such as “comprising,” “including,” “carrying,” “having,”“containing,” “involving,” and the like are to be understood to beopen-ended, i.e. to mean including but not limited to. Only thetransitional phrases “consisting of” and “consisting essentially of”shall be closed or semi-closed transitional phrases, respectively, asset forth in the United States Patent Office Manual of Patent ExaminingProcedures, Section 2111.03.

What is claimed is:
 1. A method for identifying one or more tools fordetermining ovarian cancer, comprising: partitioning blood serum from afirst subject having ovarian cancer in at least a first phase and asecond phase of a first aqueous multi-phase system, wherein the secondphase is immiscible with the first phase at equilibrium; determining arelative measure of interaction with the first and second phases of thefirst system, of a species within the blood serum selected from thegroup consisting of EGFR, IL-8, G-CSF, sIL-6R, IGFBP-1, bFGF, andRANTES; determining a relative measure of interaction of the specieswith the first and second phases of the first system; determining adifference in the relative measure of interaction of the species of thefirst subject with the first and second phases of the first system,versus the relative measure of interaction of the species of the secondsubject with the first and second phases of the first system; and basedupon the difference, selecting the first system as a tool fordetermining ovarian cancer in a subject based upon the determination ofthe relative measures of interaction of the species.
 2. The method ofclaim 1, further comprising determining a difference in the relativemeasure of interaction of the species of the first subject versus therelative measure of interaction of the species of the second subject, inboth the first system and a different, second aqueous multi-phasesystem.
 3. The method of claim 1, comprising determining a difference inthe relative measure of interaction of the species of the first subjectversus the relative measure of interaction of the species of the secondsubject, in only a single aqueous multi-phase system.
 4. The method ofclaim 1, further comprising: determining a relative measure ofinteraction of the species of the first subject, with at least a firstphase and a second phase of a second aqueous multi- phase system;determining a relative measure of interaction of the species of thesecond subject, with the first and second phases of the second system;determining a difference in the relative measure of interaction of thespecies of the first subject with the first and second phases of thesecond system, versus the relative measure of interaction of the speciesof the second subject with the first and second phases of the secondsystem; and based upon the difference, selecting the second system as atool, in conjunction with the first system, for determining ovariancancer.
 5. The method of claim 1, further comprising: determining arelative measure of interaction of the species of the first subject,with at least a first phase and a second phase of a third aqueous multi-phase system; determining a relative measure of interaction of thespecies of the second subject, with the first and second phases of thethird system; determining a lack of significant difference in therelative measure of interaction of the species of the first subject withthe first and second phases of the third system, versus the relativemeasure of interaction of the species of the second subject with thefirst and second phases of the third system; and based upon the lack ofsignificant difference, rejecting the third system as a tool fordetermining ovarian cancer.
 6. The method of claim 1, wherein the firstaqueous multi-phase system comprises a polymer.
 7. The method of claim1, wherein the first aqueous multi-phase system comprises a salt.
 8. Themethod of claim 1, wherein the first aqueous multi-phase systemcomprises a surfactant.
 9. The method of claim 1, wherein the relativemeasure of interaction comprises a partition coefficient.
 10. The methodof claim 1, wherein the first subject is human.
 11. A method ofdetermining ovarian cancer, comprising: partitioning blood serum from asubject in at least a first phase and a second phase of a first aqueousmulti-phase system, wherein the second phase is immiscible with thefirst phase at equilibrium; determining a relative measure ofinteraction of a species within the blood serum selected from the groupconsisting of EGFR, IL-8, G-CSF, sIL-6R, IGFBP-1, bFGF, and RANTES withthe first and second phases of the first system; and from the process ofdetermining the relative measure of interaction of the species with thefirst and second phases of the first system, determining whether thesubject has ovarian cancer.
 12. The method of claim 11, comprisingdetermining whether the subject has ovarian or has an elevated risk ofovarian cancer.
 13. The method of claim 11, comprising determiningmalignancy of the ovarian cancer in the subject.
 14. The method of claim11, comprising determining a response to treatment of the ovarian cancerof the subject.
 15. The method of claim 11, comprising determining ametastatic state of the ovarian cancer.
 16. The method of claim 11,comprising determining an aggressiveness potential of the ovariancancer.
 17. The method of claim 11, wherein the first aqueousmulti-phase system comprises a polymer.
 18. The method of claim 11,wherein the first aqueous multi-phase system comprises a salt.
 19. Themethod of claim 11, wherein the first aqueous multi-phase systemcomprises a surfactant.
 20. The method of claim 11, wherein the relativemeasure of interaction comprises a partition coefficient.